System, method and device for tissue-based diagnosis

ABSTRACT

The current invention provides devices, methods and systems involving application of energy and/or a liquefaction promoting medium to a tissue of interest to generate a liquefied sample comprising tissue constituents so as to provide for rapid tissue sampling, tissue decontamination as well as qualitative and/or quantitative detection of analytes that may be part of tissue constituents (e.g., several types of biomolecules, drugs, and microbes). In addition, the current invention provides specific compositions of the said liquefaction promoting medium so as to facilitate liquefaction, preserve liquefied tissue constituents, and enable delivery of molecules into tissues. Determination of tissue composition in the liquefied tissue sample can be used in a variety of applications, including diagnosis or prognosis of local as well as systemic diseases, evaluating bioavailability of therapeutics in different tissues following drug administration, forensic detection of drugs-of-abuse, evaluating changes in the tissue microenvironment following exposure to a harmful agent, and various other applications. The methods, devices and systems are used to deliver one or more drugs through or into the site of the tissue to be liquefied.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/152585, filed Feb. 13, 2009, which is incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

The biomolecular composition of human tissues, represented by amultitude of lipids, proteins, nucleic acids, and other miscellaneousmolecules, is a sensitive indicator of local pathologies, such ascancer, allergies, and eczema, as well as several systemic diseases,such as cardiovascular disease, Alzheimer's disease, and diabetes. Inaddition, tissue molecular composition also holds critical informationabout the body's exposure to exogenous chemical and biological entities.However, this information is not currently used in diagnostic methodsdue to a lack of patient-friendly and standardized methods for routinesample collection from tissues. Instead, clinical diagnosis isinvariably performed by visual observation and histopathologicalanalysis of tissue biopsies, which are highly limited due to theirqualitative nature, leading to increased misdiagnosis and inappropriateuse. In addition to being invasive, current methods also fall short inexplaining a complete molecular genesis of diseases, and fail todistinguish between diseases.

Prior approaches using physical and chemical methods for assessingtissue fluid have focused chiefly on extracting a few low molecularweight molecules that are freely present in the interstitial fluid, suchas calcium and glucose. Use of tape stripping for physically harvestingsuperficially-lying tissue constituents with an adhesive tape has beenreported; however this technique has been shown to be limited byinefficacy, lack of a standardized protocol, and high heterogeneity intissue sampling.

BRIEF SUMMARY OF THE INVENTION

The current invention describes system, method and device, as well ascompositions useful in such systems, methods and devices, involvingapplication of energy to a tissue of interest to generate a liquefiedsample comprising tissue constituents so as to provide for rapid tissuesampling, as well as qualitative and/or quantitative detection ofanalytes that may be part of tissue constituents (e.g., several types ofbiomolecules, drugs, and microbes). Determination of tissue compositioncan be used in a variety of applications, including diagnosis orprognosis of diseases, evaluating bioavailability of therapeutics indifferent tissues following drug administration, forensic detection ofdrugs-of-abuse, evaluating changes in the tissue microenvironmentfollowing exposure to a harmful agent, tissue decontamination andvarious other applications.

The current invention provides methods and devices for generating aliquefied tissue sample from a subject—living or diseased. The deviceand method involve applying energy and a liquefaction promoting mediumto a tissue of interest of a subject, the applying producing a liquefiedtissue sample, and collecting the liquefied tissue sample. In someembodiments, an analysis for the presence or absence of at least oneanalyte in the liquefied tissue sample is performed, wherein theanalysis facilitates diagnosis of a condition of interest. In certainembodiments, the analysis involves generating an analyte profile fromthe liquefied tissue sample and comparing the analyte profile to areference analyte profile, wherein the comparing facilitates diagnosisof a condition of interest.

In some embodiments, the purpose of said tissue liquefaction is toremove, or decontaminate the tissue from undesired substances.Non-limiting examples of such undesired substances include chemicals,environmental contaminants, biological toxins, and in general substancesthat are considered toxic or hazardous to the body. In certainembodiments, the said method of decontamination is performed bycontinuously moving the tissue liquefaction device overtissue-of-interest until removal of undesired substances at a preferredlevel is attained.

In some embodiments, the liquefaction promoting agent comprises of oneof more of sodium chloride, potassium chloride, sodium phosphatedibasic, potassium phosphate monobasic,3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid,N,N-bis(2-hydroxyethyl)glycine, tris (hydroxymethyl) methylamine,N-tris(hydroxymethyl)methylglycine,4-2-hydroxyethyl-1-piperazineethanesulfonic acid,2-{[tris(hydroxymethypmethyl]amino}ethanesulfonic acid,3-(N-morpholino)propanesulfonic acid,piperazine-N,N′-bis(2-ethanesulfonic acid), dimethylarsinic acid, salinesodium citrate, 2-(N-morpholino)ethanesulfonic acid. In certainembodiments, the liquefaction promoting agent comprises of one or moreof a protease inhibitor, an RNase inhibitor, or a DNase inhibitor. Incertain embodiments, the liquefaction promoting agent comprises at leastone of free radical scavenger, a defoaming agent, and a proteinstabilizer. In certain embodiments, the liquefaction promoting agentcomprises at least one of Brij-30, 3-(Decyl dimethyl ammonio) propanesulfonate (DPS), 3-(Dodecyl dimethyl ammonio) propane sulfonate (DDPS),N-lauroyl sarcosine (NLS), Triton X-100, Sodium Dodecyl Sulfate, DMSO,fatty acids, azone, EDTA, or sodium hydroxide. In certain embodiments,the liquefaction promoting agent comprises a suspension of abrasiveparticles. In certain embodiments, the abrasive particles comprisesilica or aluminum oxide.

In some embodiments, the energy is applied in the form of ultrasound,mechanical, optical, thermal, or electrical energy. In certainembodiments, the mechanical energy is applied by an abrasive material.In certain embodiments, the thermal energy is applied in the form ofradio frequency energy. In certain embodiments, the optical energy isapplied in the form of a laser.

In some embodiments, the liquefied tissue sample is generated for eachof a healthy tissue of interest of the subject and a suspected diseasedtissue of interest of the subject, and the analysis comprises comparinganalytical results from the healthy tissue sample with analyticalresults from the suspected diseased tissue sample, wherein the comparingfacilitates diagnosis of a condition of interest. In some embodiments,the liquefied tissue sample is generated for multiple tissue sites andthe analysis comprises comparing analytical results from the multipletissue sites, wherein said comparing facilitates diagnosis of acondition of interest. In some embodiments, the liquefied tissue sampleis collected from multiple tissue sites, and the samples are combined tomake a diagnosis.

In some embodiments, the liquefied tissue sample is collected byaspiration. In certain embodiments, the collecting is by retaining theliquefaction agent in a housing placed in contact with the tissue. Incertain embodiments, the collecting is by mechanized transfer of theliquefied tissue sample in a housing located in the device.

In some embodiments, the liquefied tissue sample is mixed with asubstance which assists in further liquefaction and in stabilization ofanalytes of interest for storage or transportation. In certainembodiments, the transferred tissue sample from that sample container ismixed with the substances which are pre-stored in a container. Examplesinclude a protein stabilizer such as protease inhibitor, a nucleic-acidstabilizer such as EDTA, phenol, nonspecific proteinase, an RNaseinhibitor and a DNase inhibitor, a defoaming agent, and surfactants suchas Triton X-100, Sodium Dodecyl Sulfate, and DMSO, and abrasiveparticles comprise silica or aluminum oxide.

In certain embodiments, the device evaluates the tissue of interestprior, during, or after liquefaction process. In certain embodiments,the evaluation is performed by electrochemical, biochemical, or opticalmeans. In some embodiments, the evaluation involves measurement oftissue's electrical conductivity. In an exemplary embodiment, electricalconductivity is measured by a means applying an AC electrical signalacross the tissue of interest. The said electrical signal has voltagebetween 0.1 mV and 10 V and frequency between 1 Hz and 100 kHz.

In some embodiments, the device involves detecting certain tissueconstituents in the liquefied tissue sample prior to analysis of ananalyte of interest, such as a disease marker. In certain embodiments,the detecting is by electrochemical, biochemical, or optical means. Insome embodiments the electrochemical means of detecting is anion-elective electrode. In some embodiments the optical means ofdetecting is measuring the absorption or scattering coefficient of aliquid solution.

In some embodiments, the energy is applied to a tissue in the form ofultrasound with a mechanical index between 0.1 and 50. In certainembodiments, the energy is applied by contacting the tissue with amoving abrasive surface. In certain embodiments, the energy is appliedto the tissue by contacting the tissue with a moving brushing devicecomprising a plurality of bristles. In certain embodiments, the energyis applied to the tissue by mechanical insertion of a patch bearingplurality of micro-needles into the tissue; and further injection ofliquefaction medium through the micro-needles into the tissue. In someembodiments, additional energy is applied by moving the saidmicro-needle patch after its insertion into the tissue. In certainembodiments, the energy is applied to the tissue by mechanized stirringof the liquefaction agent. In certain embodiments, the energy is appliedto the tissue by contacting the tissue with a high velocity jetcomprising of liquefaction promoting medium, which may also containabrasive particles in different embodiments.

In some embodiments, the tissue comprises breast, prostate, eye, vagina,bladder, nail, hair, colon, testicles, or intestine. In certainembodiments, the tissue comprises skin or a mucosal membrane. In certainembodiments, the tissue comprises lung, brain, pancreas, liver, heart,bone, or aorta wall.

In some embodiments, the analyte comprises a small molecule, a drug ormetabolite thereof, a polypeptide, a lipid, a nucleic acid, or amicrobe. In certain embodiments, the analyte comprises an antibody, acytokine, an illicit drug, or a cancer biomarker.

In some embodiments, the liquefied tissue sample is held in a container,and the analyte profile is generated by integrating the liquid containerwith one or more analytical devices. In certain embodiments, the tissueliquefaction device contains a means for measuring the concentration ofa calibrator analyte to provide a means for calibrating the analysis ofthe analyte.

In some embodiments, the device involves diagnosing allergic disease ina subject, and the device comprises means for analyzing the liquefiedtissue sample for the presence or absence of IgE and IgG antibodies,cytokines such as IL4, IL5, IL10, IL-12, IL13, IL-16, GM-CSF, RANTES,MCP-4, CTACK/CCL27, IFN-g, TNFa, CD23, CD-40, Eotaxin-2, and TARC,wherein the analysis facilitates diagnosis of allergic disease in thesubject.

In some embodiments, the device involves diagnosing cancer in a subject,and the device comprises means for analyzing the liquefied tissue samplefor the presence or absence of one or more cancer markers, wherein theanalysis facilitates diagnosis of cancer in the subject. In certainembodiments, the tissue of interest is breast, colon, prostate, skin,testicle, intestine, or mouth.

In some embodiments, the device involves diagnosing heart disease in asubject, and the device comprises means for analyzing the liquefiedtissue sample for the presence or absence of one or more of cholesterol,triglycerides, lipoproteins, free fatty acids, and ceramides, whereinthe analysis facilitates diagnosis of heart disease in the subject.

In some embodiments, the device involves detecting the presence of anillicit drug, or metabolite thereof, in a subject, and the devicecomprises means for analyzing the liquefied tissue sample for thepresence or absence of an illicit drug, or metabolites thereof, whereinthe analysis provides for detection of illicit drugs in the subject.

In some embodiments, the device involves detecting a microorganism in asubject, and the device comprises means for applying energy and aliquefaction medium to a tissue of interest in a subject and analyzingthe liquefaction medium for the presence or absence of a microorganism,wherein the analysis provides for detection of the presence or absenceof a microorganism.

Another object of the current invention is to provide a method anddevice for liquefying a tissue of a subject for facilitating the passageof a drug across or into the tissue. The method and device disclosedabove are applicable not only to collection of tissue constituents butalso to drug delivery. The device and method involve applying energy anda liquefaction medium to a tissue of interest of a subject, anddelivering a drug through or into the site of the tissue to beliquefied. The advantage of using the present invention is 1) to providehigher fluxes of drugs into a tissue, and 2) to allow greater control offluxes into a tissue. Drugs which would simply not pass through thetissues such as the skin are forced through the tissues when the methodis applied.

In some embodiments, the present invention offers a method fordelivering one or more drugs through the tissue to be liquefied into thecirculatory system, which circumvents degradation in thegastrointestinal tract and rapid metabolism by the liver from whichdrugs to be routinely administered either orally or by injection suffer.In certain embodiments, the current invention provides a method anddevice for delivering one or more drugs locally to the tissue ofinterest, thus limiting side effects to the healthy tissues. The methodand device may also be applicable for enhancing transport to cellularmembranes.

In particular, the device of the present invention consists of thefollowing major components: 1) an energy generator; 2) a liquefactionpromoting medium; 3) a reservoir to hold drugs to be delivered and/orcollect the liquefied tissue sample.

A drug to be administered can be added into the liquefaction mediumprior or during tissue liquefaction process. In an alternate embodiment,application of energy is in combination of the liquefaction medium whichdoes not contain a drug can be used for liquefying a tissue, andsubsequently a drug in an appropriate carrier such as a patch can beapplied on a site of the tissue to be liquefied.

The transport of drug into the tissue can be further enhanced by thesimultaneous or subsequent application of a secondary driving force suchas chemical permeability or transport enhancers, convection, osmoticpressure gradient, concentration gradient, iontophoresis,electroporation, magnetic field, ultrasound, or mechanical pressure. Thedriving force can be applied continuously over a period of time or atintervals during the period of liquefaction.

In some embodiments, the tissue to be administered comprises an organsas well as biological surfaces. In certain embodiment, the biologicalsurfaces comprise a biological membrane and cellular membrane. Incertain embodiment, the biological membrane comprises skin or a mucosalmembrane. In certain embodiments, the biological membrane comprises abuccal membrane, eye, vagina, colon, or intestine. In some embodiment,the tissue comprises a diseased tissue.

In one embodiment, a device is provided that can be used on a tissue toobtain a liquefied sample comprising an energy source operably coupledto the tissue, and a chamber, operably coupled to said tissue, capableof delivering liquefaction promoting medium to and/or collecting saidliquefied sample from said tissue.

In another embodiment, the device can be used on a tissue which is apart of a living organism; and the tissue can be excised from theorganism prior to diagnosis.

In another embodiment, the device of claim 1 wherein the liquefiedtissue sample is transferred to an assay for monitoring the presence orabsence of at least one analyte.

In yet another embodiment, the chamber of the device can be asponge-bellow assembly where the sponge is capable of storing saidliquefaction promoting medium and/or liquefied tissue sample.

In another embodiment, a device is provided comprising an energy sourceoperably coupled to the tissue, and a chamber, operably coupled to saidtissue, capable of delivering liquefaction promoting medium to and/orcollecting said liquefied sample from said tissue; also comprises atube/needle, connected to said chamber, capable of delivering theliquefaction promoting medium to and/or aspirating liquefied tissuesample from the tissue.

In still another embodiment, a device is provided comprising an energysource operably coupled to the tissue, and a chamber, operably coupledto said tissue, capable of delivering liquefaction promoting medium toand/or collecting said liquefied sample from said tissue; also comprisesa sample container, operably connected to said chamber, capable ofstoring aspirated liquefied tissue sample containing analytes, ortransferring said aspirated liquefied tissue sample to an ancillarychamber; wherein the chamber is used only to deliver the liquefactionpromoting medium to the chamber.

In another embodiment, a pressurized container and/or vacuum containeris part of the device, which facilitates transfer of said liquefactionpromoting medium and/or liquefied tissue sample.

In one embodiment, the energy emitted from the energy source in thedeice is in the form of ultrasound, mechanical, optical, thermal, orelectrical energy. In a particular embodiment, the mechanical energy isapplied to the tissue by an abrasive material, vacuum, pressure or shearforce. In another embodiment, the thermal energy is applied to thetissue in the form of radio frequency energy. In another embodiment, theoptical energy is applied to the tissue in the form of a laser.

In yet another embodiment, a device is provided comprising an energysource operably coupled to the tissue, and a chamber, operably coupledto said tissue, capable of delivering liquefaction promoting medium toand/or collecting said liquefied sample from said tissue furthercomprising a sample container, operably connected to said chamber,capable of storing aspirated liquefied tissue sample containinganalytes, or transferring said aspirated liquefied tissue sample to anancillary chamber; wherein the chamber is used only to deliver theliquefaction promoting medium to the chamber.

In another embodiment, a device is provided comprising an energy sourceoperably coupled to the tissue, and a chamber, operably coupled to saidtissue, capable of delivering liquefaction promoting medium to and/orcollecting said liquefied sample from said tissue, wherein the energysource comprises of a pad connected to a shaft.

In a more particular embodiment, the shaft has a pressure sensing unit,which maintains a predetermined pressure profile on to the tissue uponcontact.

In another embodiment, the pad is selected from a group consisting of anabrasive surface and a patch comprising of a plurality of micro-needles.

In yet another embodiment, the device further comprises a plunger,operably connected to the top of the chamber.

In another embodiment, the device is divided into an upper and lowerunit, and wherein the lower unit is detachable from said upper unit;wherein the upper unit comprises the energy source and the lower unitcomprises the chamber.

In still another embodiment, the device further comprises an analyticalunit operably connected to the chamber, and where the analytic unit iscapable of performing temporal monitoring of the tissue sample byelectrochemical, biochemical or optical means; or the analytic unit iscapable of analyzing the analytes within said liquefied tissue sample.

In another embodiment, the device is connected to a diagnostic probe ora catheter; wherein the diagnostic probe is selected from a groupconsisting of endoscope, colonoscope, and laparoscope.

In still another embodiment, the use of the device results in situliquefaction of the tissue sample.

In another embodiment, the device contains a liquefaction promotingmedium that can preserve and enhance the detection of proteins, lipidsand nucleic acids, comprising: 3-(decyl dimethyl ammonio) propanesulfonate (DPS) and polyethylene glycol dodecyl ether (Brij 30)dissolved in a buffered solution; and where the concentration of3-(decyl dimethyl ammonio) propane sulfonate and polyethylene glycoldodecyl ether (B30) is between 0.01-10% (w/v); and where the 3-(decyldimethyl ammonio) propane sulfonate and polyethylene glycol dodecylether are present at a ratio of 50:50.

In yet another embodiment, the liquefaction promoting medium within thedevice is buffered in a solution comprising either phosphate-bufferedsaline, Tris-buffered saline, Tris-HCL or EDTA.

In another embodiment, liquefaction promoting medium within the devicecomprises a nonionic surfactant selected from a Brij series surfactant,a Triton-X surfactant, and a Sorbitan surfactant; an anionic or azwitterionic surfactant; and a hydrophilic solvent; wherein the mediumhas a total concentration of the surfactants from about 0.01%-10% (w/v).

These and other features of the invention will become apparent to thosepersons skilled in the art upon reading the details of the system,method and device for tissue-based diagnosis as more fully describedbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures:

FIG. 1 (Panels a-g) is a collection of cross-sectional drawingsillustrating structure, components and functioning of various abrasiveenergy-based tissue liquefaction devices. Panels a-c and Panels e-g showthe sequential working of two separate liquefaction devices. Panel d isa schematic representation of a pressure-sensitive motorized shaftbearing an abrasive head.

FIG. 2 (Panels a-b) is a collection of cross-sectional drawings ofmoveable tissue liquefaction devices for continuous sampling of a largearea of tissues.

FIG. 3 (Panels a-c) is a collection of cross-sectional drawingsillustrating structure and components of various linear abrasivemotion-based tissue liquefaction devices. Panel c is a schematicrepresentation of a pressure-sensitive support shaft bearing a gear.

FIG. 4 (Panel a-g) is a collection of cross-sectional drawingsillustrating several types of abrasive heads.

FIG. 5 (Panels a-d) is a collection of cross-sectional device drawingsand schematics for measuring tissue's electrical conductivity.

FIG. 6 (Panels a-g) is a collection of cross-sectional drawingsillustrating structure, components and functioning of variousmicroneedle-based tissue liquefaction devices.

FIG. 7 (Panels a-e) is a collection of cross-sectional drawings of anexemplary abrasive energy-based tissue liquefaction device. Panel ashows various assembly components of the device. Panel b-d showsequential working steps of the device including transfer of theliquefaction medium to be placed in contact with the tissue (Pane b-c),sample generation by liquefaction (Panel c), and collection of thesample in a container (panel d). Panel e shows post-liquefactionretrieval of sampling container from the device.

FIG. 8 (Panels a-d) is a collection of cross-sectional drawingsillustrating sequential working steps of an exemplary microneedle-basedtissue liquefaction device: transfer of the liquefaction medium to beplaced in contact with the tissue (Pane a-b); sample generation byliquefaction (Panel c); and collection of the sample in a container(panel d).

FIG. 9 (Panels a-d) is a collection of drawings illustrating a samplingcontainer. Panels a-d show the sequential working steps for transportingand/or analysis of the generated samples. Panel a shows substrates whichselectively bind to analytes of interest are coated on the insidesurface of the container. The analytes in the liquefied tissue samplesare selectively captured by the coated substrates (Panel b). Uponsufficient incubation of the tissue sample, the sample is discardedwhile the analytes are held in the container (Panel c). The analytes areeluted by a buffer for subsequent analysis (Panel d).

FIG. 10 (Panels a-c) is a collection of drawings illustrating thescreening methodology for identifying unique surfactant formulations ofLPMs. Panel a ranks over 150 surfactant formulations in their ability topreserve protein bioactivity. Panel b ranks best formulations from Panela on their tissue solubilization potential. Panel c compares the bestLPM from entire screening—0.5% (w/v) DPS-Brij30 with other conventionalsurfactants in their potential to sample functional proteins from skintissue.

FIG. 11 (Panel a-b) is a collection of drawings illustratingLPM-assisted preservation of bioactivities of various proteins(IgE—panel a; IgE, LDH and β-gal—panel b) under mechanical stress ofultrasound exposure.

FIG. 12 (Panel a-c) is a collection of drawings illustrating the abilityof ultrasonic exposure in the presence of LPM (saline solution of 0.5%(w/v) DPS-B30) to sample a variety of functional disease biomarkers(IgE—Panel a; Cholesterol—Panel b; Bacteria—Panel c) from skin tissue.

FIG. 13 is a graph illustrating the effect of buffers in LPMs on thecompatibility with quantitative PCR.

FIG. 14 is a graph illustrating the influence of surfactant mixture onthe compatibility with quantitative PCR.

FIG. 15 is a graph illustrating the effect of ultrasound intensity andexposure time on E. Coli viability. Samples were exposed to ultrasoundat intensities of 1.7 W/cm² (•) and 2.4 W/cm² (▪). Each point representsthe mean value from three independent samples.

FIG. 16 is a photograph of agarose gel-electrophoresis of genomic DNAfrom E. coli cells sonicated at different conditions in tris-HCl. Lane 1molecular standard; lane 2 Non-treated cell; lane 3 1.7 W/cm², 2 min;lane 4 1.7 W/cm² 3 min; lane 5 2.4 W/cm², 3 min.

FIG. 17 (Panels a and b) is a graph illustrating the number of bacteriasampled by ultrasound coupling with tris-HCl, swabbing, and surfactantscrub technique, measured by (a) culture assay and (b) quantitative PCR.Each point represents the mean value from five independent samples.

FIG. 18 is a graph illustrating the effect of adding various sensitivityenhancers in LPM for enhanced detection of a model analyte—human IgEantibody, in it. Sensitivity enhancers used in the analysis are amixture of 10% w/v BSA and 0.5% w/v Tween 20 in phosphate-bufferedsaline (PBS) (open diamond); and a mixture of 10% w/v BSA and 0.5% w/vTween 20 in tris-buffered saline (closed circle). Prior to analysis,each of the sensitivity enhancers was diluted at 1:10 ratio with LPMcontaining model analyte. As a control, LPM containing model analyte(open square) and a commonly-used analytical solvent comprising of amixture of 1% w/v BSA and 0.05% w/v Tween 20 in tris-buffered saline(solid square) were used. The LPM was composed of a solution of 1% w/vmixture of NLS and Brij 30 in PBS. Error bars indicate the standarddeviation.

FIG. 19 (Panels a-b) is a collection of graphs illustrating delivery ofInulin across and of Acyclovir into pig skin in vitro after ultrasoundapplication (a) or abrasion with a plurality of bristles (b).

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Energy” as used herein means any appropriate energy that can be appliedto tissue to accomplish the objective of the methods disclosed herein(e.g., liquefying tissue). Exemplary types of energy include mechanicalenergy (e.g., abrasion, shear, vacuum, pressure, suction), ultrasound,optical (e.g., laser), magnetic, thermal, and electrical energy.

An “analyte” as used herein means any biomolecule (e.g., polypeptide,nucleic acid, lipid, and the like), drug (e.g., therapeutic drugs,drugs-of-abuse, and the like), small molecule (e.g., naturalmoisturizing factors, nicotine, and the like, with the understandingthat small molecules can also be drugs), warfare agent, environmentalcontaminant (e.g., pesticides, etc.), microbe (e.g., bacterium, virus,fungus, yeast, and the like) and the like that is present in or on thetissue and can be extracted from the tissue of interest (e.g., skin, amucosal membrane, and the like) and detected, analyzed, and/orquantified.

The term “liquefaction” is used to describe the process by which tissueand/or tissue constituents are converted to a sufficiently soluble statethrough exposure to sufficient energy and, optionally, a liquefactionpromoting medium, and can involve conversion of at least a portion of atissue structure of interest to a liquid form. A tissue sample that hasbeen subjected to liquefaction as sometimes referred to herein as a“liquefied” sample.

The term “liquefaction-promoting medium” (LPM) is used to describe asubstance that facilitates solubilization of one or more tissueconstituents, facilitates conversion of at least a portion of a tissuestructure into a liquid when exposed to energy, and/or facilitatespreservation of bioactivity of one or more solubilized tissueconstituents.

The term “liquefaction-promoting agent” (LPA) is used to describe acomponent of the liquefaction promoting medium, particularly an agentthat promotes at least solubilization and/or preservation of bioactivityof one or more tissue constituents, and/or analysis of subsequentdiagnostic assays.

A “calibration analyte” as used herein means any molecule naturallypresent in a tissue of interest at a known concentration, which canserve as a reference analyte (e.g., as a positive control to ensure adesired degree of liquefaction was achieved).

A “biomolecule” as used herein means any molecule or ion which has abiological origin or function. Non-limiting examples of biomoleculesinclude proteins (e.g., disease biomarkers such as cancer biomarkers,antibodies: IgE, IgG, IgA, IgD, or IgM, and the like), peptides, lipids(e.g., cholesterol, ceramides, or fatty acids), nucleic acids (RNA andDNA), small molecules (e.g., glucose, urea, creatine), small moleculedrugs or metabolites thereof, microbes, inorganic molecules, elements,or ions (e.g., iron, Ca2+, K+, Na+, and the like). In some embodiments,the biomolecule is other than glucose and/or is other than a cancermarker.

The term “abused drug” or “drug-of-abuse” or “illicit drug” are usedinterchangeably herein to refer to any substance which is regulated by agovernmental (e.g. federally or state regulated) of which presence in ahuman tissue, and/or presence above a certain level in a human tissue,is illegal or can be harmful to a human being. Examples of abused drugsinclude: cocaine, heroin, methyl amphetamine, and prescription drugstaken in excess of dosage, or taken without a prescription (e.g.,painkillers such as opioids).

The term “warfare agent” as used herein refers to any molecule,compound, or composition of either biological or chemical origin thatmay be used as a weapon. Examples of warfare agents include nerve gases(e.g. VX, Sarin), phosgene, toxins, spores (e.g., anthrax), and thelike.

The term “environmental contaminant” as used herein includes anymolecule, compound, or composition which can be detrimental to anindividual, e.g., when at concentrations elevated above a riskthreshold. Examples include water pollutants (e.g., fertilizers,pesticides, fungicides, insecticides, herbicides, heavy metals,halides), soil pollutants (e.g., fertilizers, pesticides, fungicides,insecticides, herbicides, heavy metals, halides), air pollutants (e.g.,NOx, SOx, greenhouse gases, persistent organic pollutants (POPs),particulate matter, smog).

The term “decontamination” as used herein includes removal from tissuesof any unwanted or undesired molecule, compound, or composition whichcan be detrimental to an individual. Examples include environmentalcontaminants (as defined above), toxic chemicals, and biological toxins.

The term “natural moisturizing factor” (NMFs) as used herein means anyone of several types of small molecules, including but not limited tofree amino acids, lactate, and urea, which are derivatives of fillagrin.NMFs can be used as analytes to facilitate assessment of general skinhealth (e.g., dry skin, flaky skin, normal skin, etc.). The term“mechanical index” as used herein means the ratio of the amplitude ofpeak negative pressure in an ultrasonic field and the square-root of theultrasound frequency (Mechanical Index=(Pressure (MPa))/(Frequency(MHz))̂0.5.

The term “drug delivery” as used herein means the delivery of one ormore drugs into blood, lymph, interstitial fluid, a cell or tissue.

The term “sensitivity enhancer” as used herein means a substance or amixture of substances that is mixed with LPM to stabilize liquefiedtissue analytes and facilitate their analysis in terms of enhancing thesensitivity and specificity of the diagnostic analytical tests.

The term “blocking reagent” is used to describe a component which isused to prevent non specific binding of analytes to substrates used in adiagnostic assay.

Before the present invention and specific exemplary embodiments of theinvention are described, it is to be understood that this invention isnot limited to particular embodiments described, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither, or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “atissue” includes a plurality of such tissues and reference to “theliquid” includes reference to one or more liquids, and so forth. It isfurther noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only,” and thelike, in connection with the recitation of claim elements, or use of a“negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

The current invention provides systems, methods and devices, as well ascompositions useful in such systems, methods and devices, involvingapplication of energy to a tissue of interest to generate a liquefiedsample comprising tissue constituents so as to provide for rapid tissuesampling, as well as qualitative and/or quantitative detection ofanalytes that may be part of tissue constituents (e.g., several types ofbiomolecules, drugs, and microbes—might want a paragraph to formallydefined what you mean by tissue constituents). Determination of tissuecomposition or constituents can be used in a variety of applications,including diagnosis or prognosis of local as well as systemic diseases,evaluating bioavailability of therapeutics in different tissuesfollowing drug administration, forensic detection of drugs-of-abuse,evaluating changes in the tissue microenvironment following exposure toa harmful agent, decontamination, and various other applications.

Another object of the current invention is to provide a method anddevice for liquefying a tissue of a subject for facilitating the passageof a drug across or into the tissue. The method and device disclosedabove are applicable not only to collection of tissue constituents butalso to drug delivery. The device and method involve applying energy anda liquefaction medium to a tissue of interest of a subject, anddelivering a drug through or into the site of the tissue to beliquefied. The advantage of using the present invention is 1) to providehigher fluxes of drugs into a tissue, and 2) to allow greater control offluxes into a tissue. Drugs which would simply not pass through thetissues such as the skin and into the circulatory system are forcedthrough the tissues when the method is applied.

Although the present invention may be described in conjunction withhuman applications, veterinary applications are within the contemplationand the scope of the present invention.

Tissue Diagnostics Energy Application Devices

The tissue liquefaction devices disclosed herein can be generallydescribed as having an energy source/generator operably coupled to areservoir unit/housing, where the reservoir houses a medium in whichanalytes are collected and which, in most embodiments, facilitatestransfer of energy to the tissue of interest and can thus, wheredesired, facilitate liquefaction of a tissue sample. In use, thereservoir housing is placed in contact with the subject's tissue to makecontact between the medium and the tissue, and the energy source isactivated. The device can be operably coupled to additional energysources, (e.g., abrasive actuator, piezoelectric transducer, suction orpressure), which can also be applied to the tissue to facilitatetransfer of energy to the tissue. As energy is applied to the tissue,constituents of the tissue are solubilized by the energy and collectedin the medium. The medium can be retained in the reservoir housing, oralternatively be transferred to a separate container. The reservoirhousing or container can be operably coupled to a detection device thatcan quantitatively measure the tissue constituents present in themedium.

Energy can be applied to the tissue from a single energy source or as acombination of sources. Exemplary energy sources include mechanical(e.g., abrasion, shear, vacuum, pressure, and the like), piezoelectrictransducer, ultrasound, optical (e.g., laser), thermal, and electricalenergy. The intensity of the energy applied, as well as the duration ofthe energy application, may be appropriately adjusted for the particulartissue of interest and the particular application of the method. Theenergy intensity and duration of application may also be appropriatelyadjusted based on the particular liquefaction promoting medium (LPM)used in connection with the energy. In some embodiments, an energyexposure time of greater than 1 minute, greater than 90 seconds, orgreater than 2 minutes is provided in order to produce a suitableliquefied tissue sample. The magnitude of energy depends on the analyteof interest and the selection of LPM. Higher energies are required toliquefy tissues in the absence of surfactants or particles in the LPM.Use of high energies is limited by their adverse effects on the tissueor its constituents. A significant adverse effect is injurious tissuedamage. In some embodiments, therefore, it might be necessary toincorporate certain device components that provide temporal monitoring(ideally, in real-time) of the change in tissue properties or the extentof tissue liquefaction such that, once safe limit for energy exposure isreached, the device can be stopped. The temporal evaluation can beperformed prior, during, and after liquefaction process. In certainembodiments, the temporal evaluation is performed by electrochemical(e.g., tissue's electrical conductivity, measurement of certain ions byion-selective electrodes, etc), biochemical (e.g., measurement ofcertain tissue components in the LPM by enzymatic assays such as ELISAand the like), or optical (e.g., measurement of LPM turbidity byspectrophotometer, etc) means. In an exemplary embodiment, tissue'stemporal electrical conductivity is measured by applying a pre-definedAC electrical voltage across the tissue with a signal generator, andanalyzing the resultant electrical current by a multimeter. Anothersignificant adverse effect of high energy exposure is attributed totemperature elevation in the tissue, also known as thermal effects. Insome embodiments, therefore, it might be necessary to incorporate atemperature sensing element (e.g., a thermocouple) that allowsmonitoring of the temperature of the tissue and/or the LPM, facilitatingthe judgment of a safe amount of energy exposure to the tissue.

The necessary energy level is significantly reduced by appropriateselection of LPM. For example, use of saline alone along with ultrasoundresulted in recovery of less than 0.1 mg protein per cm² of skin. On theother hand, incorporation of surfactants such as DPS, NLS and Brij-30 ata concentration of 1% w/v in LPM increased protein recovery to more than0.6 mg per cm² of skin.

In certain embodiments, use of energy to liquefy tissue may lead toreduction in biological activity of solubilized tissue constituents,necessitating selection of LPM which adequately preserve the bioactivityof tissue's molecules as well as aid tissue solubilization. For example,incorporation of one or more surfactants such as DPS, NLS and Brij-30 ata concentration of 1% w/v in LPM facilitated complete preservation ofthe bioactivity of solubilized proteins and nucleic acids underultrasonic energy exposure.

In certain embodiments, energy can be applied to a tissue using anenergy delivery chamber that includes an energy producing element. Thechamber, when placed on the tissue, will expose the tissue to the energyproducing element and allow energy to be applied to the tissue withminimal interference. Such a chamber can contain LPM and provide forcontact of the LPM with the tissue such that, upon application ofenergy, tissue constituents can be directly collected into the solution.

In certain embodiments, the energy delivery chamber containing the LPMmay also comprise a diagnostic device, for example, an analyte sensor,for detecting and, optionally, quantifying analytes that may be presentin the LPM. These diagnostic devices can serve as chemical sensors,biosensors, or can provide other measurements to form a completesampling and measurement system. An element having an internal channelfor fluid transfer can be fabricated together with a sensor to form adisposable unit. The device can also be adapted to include or beprovided as a disposable unit that provides for collection of analytesin the LPM for analysis.

Alternatively, the diagnostic element can be located elsewhere (e.g.,separate from the energy device) and the contents of the energy deliverychamber in contact with tissue can be pumped using mechanical forces,capillary forces, ultrasound, vacuum, or electroosmotic forces into asensing chamber and analyzed.

In certain embodiments, e.g., when evaluating topical formulations ordetermining pharmacological parameters, the unit can be constructed tofunction as a closed loop drug delivery unit, including drug deliverymeans, analyte recovery means, sensing means to measure the analyte, andcontrol means to provide a signal to the drug delivery means.

An example of the general operation of an energy-assisted analyte deviceis described here. A portable disposable unit is inserted into aportable or bench-top energy generator. The energy generator may alsoinclude circuitry for tissue resistance measurements, analyteconcentration measurements, and display of analyte concentrationmeasurements. The system (e.g., energy applicator and disposable unit)is placed against the tissue, and energy is applied for a certain periodof time, either alone or as a combination with other physical,mechanical, electrical, and chemical forces. The tissue of interest isliquefied, and analytes from the liquefied tissue are collected in thedisposable unit and are measured using appropriate assays.

The preferred embodiment of the present invention and its advantages arebest understood by referring to FIGS. 1 through 19 of the drawings, likenumerals being used for like and corresponding parts of the variousdrawings.

Referring to FIGS. 1 a through 1 g, the structure, components andfunctioning of abrasive energy-based tissue liquefaction devices areshown. Panels a through c of FIG. 1 show the sequential working of adevice that utilizes a rotary abrasive component 101 as means forapplying energy to tissues for liquefaction. Liquefaction is achieved byplacing and setting abrasive component 101 in motion against a tissue ofinterest 107. Abrasive component 101 is attached to a shaft 102, whichis further connected to a rotary motor 103 in the device. In someembodiments, shaft 102 is designed to sense and control the pressureapplied by abrasive component 101 on tissue 107. In an exemplaryembodiment, shaft 102 is constructed of shaft 1021 and shaft 1022 whichare connected to each other by a pressure-sensitive spring 1023 (FIG. 1d). In another embodiment, shaft 1021 and shaft 1022 sandwich betweenthem a pressure-sensing piezoelectric crystal for monitoring andcontrolling applied pressure to tissue 107. A battery pack 104 powersmotor 103, which can subsequently set abrasive component 101 in rotarymotion when directed by the device operator. Prior to liquefaction,abrasive component 101 is designed to be held in isolation againsttissue 107 using a housing 105, and specifically, a thin sheet 106located on the base of housing 105 (FIG. 1 a). Upon initiation of theliquefaction process, LPM stored in a cartridge 108 is transferred tothe housing 105 (FIG. 1 a), whereupon the LPM contacts the surface ofthe sheet material 106, followed by setting the abrasive component 101in motion against sheet 106. Material of sheet 106 is chosen such thatit can be quickly abraded by abrasive component 101, allowing LPM andabrasive component 101 to come in contact with tissue 107 leading totissue liquefaction (FIG. 1 b). Non-limiting examples of sheet 106include sheet of paper, rubber sheet, metal foil, plastic sheet, or anywater-soluble sheet. Upon completion of liquefaction process, motor 103stops and LPM containing tissue constituents is transferred to a samplecontainer 110 (FIG. 1 c) [not liquefied sample not clearly shown in 110so would need a revised figure]; or directly into a pre-vacuumizedcontainer (thus avoiding the need for suction pump 109 and container110). Where there is no pre-vacuumized container, collection of thesample is facilitated by a suction pump 109.

In some embodiments, certain device components are designed asdisposable units such that, after each use of the device, thesecomponents can be replaced to allow sterile usage. Such components mayinclude housing 105, abrasive component 101, cartridge 108, samplecontainer 110, and other fluid-handling device components, as deemednecessary to maintain device sterility. Alternatively, in someembodiments, the whole device may be made disposable.

In certain embodiments, LPM storing cartridge 108 can be replaced with asponge-bellow assembly for storage and release of LPM. Panels e throughg of FIG. 1 show the sequential working of such a device. A flexiblebellow-shaped housing 112 contains a sponge 111 filled with LPM (FIG. 1e). As the device is pushed against tissue 107, sponge-bellow housing issqueezed to release LPM and abrasive component 101 is set in motion(FIG. 1 f). Upon completion of liquefaction process, motor 103 stops andLPM containing tissue constituents is transferred to a sample container110 (FIG. 1 g). Collection of the sample is facilitated by a suctionpump 109. Alternatively, in some embodiments the suction pump 109 andcontainer 110 may be avoided by collecting the sample into the sponge bylifting the device back into its original position.

Referring to FIGS. 2 a and 2 b, the structure and components of moveabletissue liquefaction devices designed for continuous sampling of a largearea of tissue are shown. Panel a of FIG. 2 show a device that utilizesa rotary abrasive component 201 as means for applying energy to tissuesfor liquefaction. Liquefaction is achieved by placing and settingabrasive component 201 in motion against a tissue of interest 207.Abrasive component 201 is attached to a shaft 202, which is furtherconnected to a rotary motor 203 in the device. In some embodiments,shaft 202 is designed to sense and control the pressure applied byabrasive component 201 on tissue 207. In an exemplary embodiment, shaft202 is constructed of two distinct shafts which are connected to eachother by a pressure-sensitive spring or a pressure- pressure-sensingpiezoelectric crystal for monitoring and controlling the appliedpressure to tissue 207. A battery pack 204 powers motor 203, which cansubsequently set abrasive component 201 in rotary motion when directedby the device operator. Once the device is placed against tissue 207, acontinuous liquefaction procedure is initiated by performing three keyprocesses—LPM stored in a cartridge 208 is continuously delivered tohousing 212 at the device-tissue interface; abrasive component 201 isset in motion against tissue 207; and liquefied tissue sample iscontinuously collected in a sample container 210 using a suction pump209. The device can be moved around such that additional tissue surfacesare exposed to the device and liquefied. When desired, the liquefactionprocess can be stopped by switching-off motor 103 and cumulative tissuesample can be accessed from container 210.

In some embodiments, additional device components may be used forpreventing LPM leakage from housing 212 due to the motion of device overtissue surface. In an exemplary embodiment, suction pump 209 can be usedto create a vacuum-assisted seal between tissue 207 and chamber 206located in a flanged housing 205 around the device.

Panel b of FIG. 2 show a device that utilizes a piezoelectric element251 as means for applying mechanical energy to tissues for liquefaction.Piezoelectric element 251 is placed in a housing 252 that interfaceswith a tissue of interest 259, and liquefaction is achieved byactivating piezoelectric element 251 with LPM present as a couplingfluid between tissue 259 and piezoelectric element 251. Piezoelectricelement 251 is a transducer of electrical energy, which is supplied toit by means of circuitry placed in a flexible tubing 253. Duringliquefaction, LPM is supplied to housing 252 by a flexible tubing 254using an operator-controlled injection system 256. Liquefied tissuesample can be simultaneously collected from housing 252 into a samplecontainer 257 using a flexible tubing 255. Sample collection isfacilitated by a suction pump 258 which is serially connected to samplecontainer 257. In some embodiments, suction pressure created in housing252 by suction pump 258 may provide for an effective seal betweenhousing 252 and tissue 259 for preventing LPM leakage from housing 252during liquefaction. In some embodiments, suction pressure created inhousing 252 by suction pump 258 may provide for an additional source ofenergy for liquefaction.

In some embodiments, housing 252 may be moved to liquefy additionaltissue surfaces and collect a sample representing tissue constituentsaccumulated from various tissue surfaces. In such a device LPM iscontinuously supplied to housing 252 by tubing 254 and sample iscontinuously collected by tubing 255.

In certain embodiments, the device in FIG. 2 b may operate without apiezoelectric element 251. In this embodiment, the LPM which flows froma tubing 254 into the housing 252 makes contact with the tissue andliquefies the tissue. Liquefied tissue is collected from the housing bytubing 255. The housing may be moved continuously or intermittently tocollect samples from a large tissue area. The device may have additionalmeans that are practically necessary to allow the movement of the deviceon a tissue, liquefaction of tissue and collection of liquefied tissue.In certain embodiments, either pressure or vacuum but not both may beused to direct LPM towards the tissue and collect liquefied tissue.

In certain embodiments, liquefaction devices may be integrated with adiagnostic probe such as endoscope, colonoscope, laparoscope, and thelike.

Referring to FIGS. 3 a through 3 c, the structure and components ofliquefaction devices that utilize an oscillating abrasive component asmeans for applying energy to tissues for liquefaction are shown.Referring to FIGS. 3 a, liquefaction is achieved by placing and settingabrasive component 301 in motion against a tissue of interest 311.Linear motion can be achieved, for example, by a rack and pinionarrangement (FIG. 3 a). Specifically, abrasive component 301 is attachedto a rack 302, which slides in a linear oscillatory motion using acircular gear 303 (pinion). Gear 303 is driven in oscillatory circularmotion by a motor 304. A battery pack 305 powers motor 304. In someembodiments, motor 304 is a servo motor which may require an electronicmicrochip controller 306 to produce oscillatory circular motion. Priorto liquefaction, abrasive component 301 is designed to be held inisolation against tissue 311 using a housing 307, and specifically, athin sheet 308 located on the base of housing 307. LPM can be pre-storedin housing 307, for instance, so that it is in contact with 308. In someembodiments, LPM may be transferred to housing 307 from a cartridgelocated elsewhere in the device. Liquefaction process is initiated bysetting the abrasive component 301 in linear motion against sheet 308.Material of sheet 308 is chosen such that it can be quickly abraded byabrasive component 301, allowing LPM and abrasive component 301 to comein contact with tissue 311 leading to tissue liquefaction. Non-limitingexamples of sheet 311 include sheet of paper, rubber sheet, metal foil,plastic sheet, or any water-soluble sheet. Upon completion ofliquefaction process, motor 304 stops and LPM containing tissueconstituents is transferred to a sample container 309. Collection of thesample is facilitated by a suction pump 310. In certain embodiments, thesample may be directly collected in a pre-vacuumized container, avoidingthe need of suction pump 310 and container 310.

In some embodiments, certain device components are designed asdisposable units such that, after each use of the device, thesecomponents can be replaced to allow sterile usage. Such components mayinclude housing 307, abrasive component 301, sample container 309, andother fluid-handling device components, as deemed necessary to maintaindevice sterility. Alternatively, in some embodiments, the whole devicemay be made disposable.

In some embodiments, the linear oscillatory motion of abrasive component301 may be generated by other mechanism such as using linear motors,linear motion actuators, ball screw assembly, leadscrew assembly,jackscrew assembly, and other devices for translating rotational motionto linear motion.

In some embodiments, a single rack and pinion system as described inFIG. 3 a may be replaced with an arrangement of multiple gears and abelt as exemplified in FIG. 3 b. Specifically, a belt 327 (not clearwhere belt is on figure—need revised figure) is mounted on gears 321,322, 323, 324, 325 and 326. An abrasive component 328 is attached tobelt 327 and is set in a linear oscillatory motion when gear 321 isdriven by motor 304 in an oscillatory rotation motion. While gears 321,322 and 326 are fixed to the housing of device, gears 323, 324 and 325are mounted on shaft 328. Shaft 328 is fixed to the housing of device.In some embodiments, shaft 328 has a flexible length such that, asabrasive component 328 is pressed against a non-flat tissue surface,shafts 328 attached with gears 323, 324 and 325 are able to adjust theirlengths in order to make abrasive component 328 contour with thenon-flat tissue surface. Additionally, shaft 328 may be designed tosense and control the pressure applied by abrasive component 328 ontissue surface. In an exemplary embodiment, shaft 328 is constructed ofshaft 3281 and shaft 3282 which are connected to each other by apressure-sensitive spring 3283 (FIG. 3 c).

Referring to FIGS. 4 a through 4 g, several designs of abrasivecomponent used in devices, methods and systems disclosed in thisinvention are described. FIG. 4 a illustrates an abrasive componentcomprising of a sheet of abrasive material with uniform thickness.Non-limiting examples of abrasive material with uniform thicknessinclude fabric, abrasive crystals (e.g., quartz, metal, silica, siliconcarbide, dust and derivatives of aluminum (such as AlO₂), diamond dust,polymeric and natural sponge, and the like, etc. In some embodiments, itmay be advantageous to design an abrasive component with heterogeneousabrasiveness, for example, those having spatial variation ofabrasiveness. In an exemplary embodiment, abrasive component is a discwith a gradient of abrasiveness that varies from high abrasiveness atdisc's center to low abrasiveness at the disc periphery (FIG. 4 b). Insome embodiments, the shape of abrasive component may be varied to anon-planar geometry. In exemplary embodiments, FIG. 4 c shows anabrasive component with a smooth and rounded tissue-facing surface(aspect ratio—defined as the ratio of height and width—may vary from 10to 0.1), and FIG. 4 d shows a circular ring-shaped abrasive component.FIGS. 4 e through 4 g show embodiments of abrasive components usingbrush as means for tissue abrasion. FIG. 4 e illustrates an abrasivecomponent comprising of a brush with bristles of uniform height andabrasiveness. In some embodiments, abrasive component comprises of abrush with bristles of different height and/or abrasiveness. FIG. 4 fshows an exemplary embodiment of a circular disc-shaped brush withbristles of high abrasiveness at the center surrounded by bristles withlow abrasiveness in the disc periphery. FIG. 4 g shows an exemplaryembodiment of a brush with bristles of different lengths forming asmooth and rounded tissue-facing surface (aspect ratio—defined as theratio of height and width of abrasive component—may vary from 10 to0.1).

Referring to FIGS. 5 a through 5 d, device components for measuring atissue's electrical conductivity are disclosed. While high energyexposure favorably liquefies tissues, its use may lead to significantadverse effects such as injurious tissue damage. In some embodiments,therefore, it might be necessary to incorporate certain devicecomponents that provide temporal monitoring (ideally, in real-time) ofthe change in tissue properties, e.g., tissue's electrical conductivity,such that, once safe limit for energy exposure is reached, the devicecan be stopped. Temporal measurement and monitoring of tissue'selectrical conductivity during liquefaction process can be done byapplying a pre-defined AC electrical voltage across the tissue ofinterest 503 using a measurement electrode 501 placed on tissue 503 anda reference electrode 502 placed in the vicinity of the region on tissue503 that is being liquefied. The resultant electrical current across thetwo electrodes, as measured by an ammeter 504, can be taken as a measureof tissue's electrical conductivity. In some embodiments, measurementelectrode 501 is maintained in electrical contact with LPM, or directlywith the region on tissue 503 that is being liquefied. In an embodiment,measurement electrode 501 is located as an inner surface lining of LPMhousing 509 (FIG. 5 a). In certain embodiments, measurement electrode isa sliding contact 506 that is fastened to a motorized shaft 510 immersedin LPM (FIG. 5 b). Electrical current is transmitted by sliding contact506 to an isolated stud 505 secured on the device housing. In someembodiments, reference electrode 502 is an extension of LPM housing 509and is placed in peripheral vicinity of the region on tissue 503 that isbeing liquefied (FIG. 5 a and FIG. 5 b). In some embodiments, referenceelectrode is a handheld cylindrical electrode 507 that is electricallyconnected with the electrical conductivity measurement componentslocated in the liquefaction device (FIG. 5 c). In some embodiments,reference electrode is a patch electrode 508 that is electricallyconnected with the electrical conductivity measurement componentslocated in the liquefaction device (FIG. 5 d).

Referring to FIGS. 6 a through 6 g, structure, components andfunctioning of devices utilizing microneedle-based tissue liquefactionare disclosed. Microneedle-based devices apply energy to tissues throughmechanical disruption of tissue components which is primarilyaccomplished by pushing microneedles into the tissue. FIG. 6 a shows thebasic design of a microneedle patch 601 bearing a multitude ofmicroneedles 602 which are pre-filled with LPM 613. Microneedle patch601 can be inserted in the tissue of interest allowing disruption anddissolution of tissue components in LPM 613. LPM 613 can be lateraspirated from patch 601 for diagnostic analysis.

Additional energy for liquefaction may be applied by post-insertionmotion of microneedles inside the tissue. FIG. 6 b illustrates avibratory component 603 which may be secured on microneedle patch 601,which after insertion of patch 601 into tissue can be activated tovigorously shake microneedles 602 inside the tissue. Vibratory component603 contains a multitude of mechanical vibrators 6031 and abattery-operated electronic circuit board 6032 for powering andcontrolling the motion of mechanical vibrators in desired directions. Inan exemplary embodiment, mechanical vibrators 6031 can be vibrated indirections parallel and perpendicular to the axis of microneedles 602.

In some embodiments, motion of microneedles post-insertion may beproduced by the motion of each microneedle 602 with respect to patch601. FIG. 6 f discloses an electromagnet 612 placed on top of patch 601.Electromagnet 612 may be used to produce oscillatory motion of eachmicroneedle 602 along its axis. This can be achieved by fastening amagnet 611 on top of each microneedle 602, such that magnet 611 respondsto an alternating polarity profile of electromagnet 612 leading tooscillatory linear motion of microneedles 602. In certain embodimentsrotary motion of microneedles may be desired. Electromagnets 6121, 6122,6123 and 6124 are placed symmetrically around patch 601 (FIG. 6 g).Magnet 611 attached on top of each microneedle 602 responds toalternating polarity profile of electromagnet 6121, 6122, 6123 and 6124leading to rotary motion of microneedles 602.

In FIG. 6 b-6 e, additional energy for liquefaction may be furtherapplied by forced motion of LPM in tissue using active injection andwithdrawal of LPM through microneedles. A housing 604 placed in thedevice may contain a compressed air container 605 which can be utilizedto force LPM contained in patch 601 to flow inside tissue. A suctionpump 606 in housing 604 may be used to apply vacuum for withdrawing LPMfrom tissue. In some embodiments, compressed air container 605 andsuction pump 606 may be alternatively used for repeated injection andwithdrawal of LPM from tissue for enhanced liquefaction. Abattery-operated electronic circuit board 607 in housing 604 is used forpowering and controlling compressed air container 605 and suction pump606. In some embodiments, suction pump 606 may be additionally connectedto a sample container to aspirate and transfer liquefied tissue samplefrom patch 601 to the sample container. In certain embodiments, housing604 may be replaced by a flexible elastic cap 608 (see FIG. 6 d) fittedon top of patch 601. Flexible cap 608 may be repeated pushed in andpushed out, for example, by pushing with a finger, such that LPM isrepeatedly injected and withdrawn from the tissue through microneedles602.

Microneedles 602 may be coated with a substance 610 to enhance tissueliquefaction (FIG. 6 e). In some embodiments, substance 610 is anabrasive material which may help in enhanced disruption of tissueconstituents and their faster dissolution in LPM. In some embodiments,substance 610 is an enzyme which may cleave specific tissue componentssuch as extracellular matrix for enhanced tissue liquefaction. In someembodiments, substance 610 is a molecule that specifically binds totissue analytes of interest leading to enhanced recovery of the analytefrom the tissue. In an exemplary embodiment, substance 610 is anantibody.

Referring to FIG. 6, in some embodiments, certain device components maybe designed as disposable such that, after each use of the device, thesecomponents can be replaced to allow sterile usage. Such components mayinclude microneedle patch 601, microneedles 602, compressed aircontainer 605, suction pump 606 and other fluid-handling devicecomponents, as deemed necessary to maintain device sterility.Alternatively, in some embodiments, the whole device may be madedisposable.

Liquefaction-Promoting Medium (LPM)

The LPM can be designed to serve one or more of the following fourpurposes: a) it facilitates dispersion of tissues into its constituents,b) it acts as a medium to collect liquefied tissue constituents, and c)it inhibits degradation of the sampled constituents such that theirchemical or biological activity is retained (e.g., by preserving variousmolecules' structural conformation and by preserving the ability ofsampled microbes to multiply), and d) ensure compatibility to thesubsequent analytical techniques.

In general, LPM comprises a solvent, such as aqueous solutions (e.g.,Tris-HCl, phosphate buffered saline, etc) or organic (“non-aqueous”)liquids (e.g., DMSO, ethanol, and the like), which may additionallycontain a variety of liquefaction-promoting agents, including but notlimited to surfactants (non-ionic, anionic, or cationic), fatty acids,azone-like molecules, chelating agents (e.g., EDTA, etc), inorganiccompounds, and abrasive substances. “Liquefaction-promoting agent” asused herein refers to a component of a LPM which can facilitateliquefaction of a tissue sample and/or solubilization of tissueconstituents. Depending on the tissue type and the analytes of interest,constituents of the LPM can be rationally selected based on the criteriadescribed above. For example, a delicate tissue, such as mucosalmembrane, can be liquefied by a saline solution with minimal or nosurfactants, whereas keratinized tissues, such as skin, will requireadditional constituents, such as surfactants.

The liquefaction promoting agents within the LPM can comprise a varietyof suitable components including, but not limited to: water, tris-HClsaline (phosphate-buffered saline (PBS), and tris-buffered saline(TBS)), alcohols (including ethanol and isopropanol (e.g., in aconcentration range of 10-100% in aqueous solution)), abrasivesubstances, such as dust or derivatives of silica, aluminum oxide, orsilicon carbide (e.g., in a concentration range of 0.01-99% (w/v) inwater-based solution), surfactants, such as Brij (various chain lengths,e.g., Brij-30), 3-(Decyl dimethyl ammonio) propane sulfonate (DPS),3-(Dodecyl dimethyl ammonio) propane sulfonate (DDPS),N-lauroylsarcosine (NLS), Triton X-100, Sodium Dodecyl Sulfate (SDS) and SodiumLauryl Sulfate (SLS), HCO-60 surfactant, Hydroxypolyethoxydodecane,Lauroyl sarcosine, Nonoxynol, Octoxynol, Phenylsulfonate, Pluronic,Polyoleates, Sodium laurate, Sodium oleate, Sorbitan dilaurate, Sorbitandioleate, Sorbitan monolaurate, Sorbitan monooleates, Sorbitantrilaurate, Sorbitan trioleate, Span 20, Span 40, Span 85, SynperonicNP, Tweens, Sodium alkyl sulfates, and alkyl ammonium halides, (e.g., inconcentrations ranging between 0.01-20% in water-based solution), DMSO(e.g., in a concentration range of between 0.01-20% in water-basedsolution), fatty acids such as linoleic acid (e.g., in a concentrationrange of between 0.1-2% in ethanol:water (50:50), azone (e.g., in aconcentration range of 0.1-10% in ethanol:water (50:50), polyethyleneglycol (e.g., in a concentration range of 10-50% in water-basedsolution), histamine (e.g., in a concentration range of 10-100 mg/ml inwater-based solution), EDTA (e.g., in a concentration range of 1-100mM), and sodium hydroxide (e.g., in a concentration range of 1-100 mM).In some embodiments the LPM may contain surfactants other than TWEEN,CTAB, SPAN, or Sodium Alkyl Sulfate. In some embodiments, the LPM maycontain surfactants other than cationic surfactants. Where the LPMincludes a surfactant, the total concentration of the surfactant (w/v)in the LPM can range from at least 0.5%, to 10%, and can be, forexample, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, orabout 3%.

The LPM can include agents that facilitate preservation of bioactivityof an analyte of interest. For example, the LPM can contain free radicalscavengers (e.g., antioxidants (e.g., polyphenol, beta-carotene, lutein,lycopene, selenium, etc), vitamin A, vitamin C, vitamin E,alpha-tocopherol, butylated hydroxytoluene, sodium benzoate, sodiumformate, and the like); defoaming agents (e.g., silicone or non-siliconeanti-foaming agents such as dimethylpolysiloxane, hydrocarbon oil, lowfatty acid diglyceride, and the like); and shear protectants (e.g.,polyethylene glycol, polyvinyl alcohol, pluronic F68, and the like).“Bioactivity” as used in the context of an analyte refers to astructural conformation that facilitates detection (e.g., such as anepitope bound by a specific antibody or other structural feature that issensitive to denaturation), and may also include a biological activityof an analyte (e.g., enzymatic activity).

LPM of particular interest are those that contain a combination ofsurfactants that when used in connection with the devices, methods andsystems disclosed herein provides for a desired level of tissueconstituents in the LPM while providing for preservation of bioactivityof analytes in the LPM, particularly so as to provide for maintenance ofstructural conformation of an analyte (e.g., avoid denaturation of aprotein analyte).

Use of different combinations of surfactants including combination ofnonionic surfactant, zwitterionic surfactant and anionic surfactant inthe LPM may provide for both high levels of tissue constituents in theLPM and good preservation of bioactivity of an analyte contained in theLPM following use in devices, methods and systems described herein.

Non-limiting examples of non-ionic surfactants of interest include Brijseries surfactants (e.g., Polyethylene glycol dodecyl ether (Brij 30),Polyoxyethylene 23-lauryl ether (Brij 35), Polyoxyethylene 2-cetyl ether(Brij 52), Polyoxyethylene 10-cetyl ether (Brij 56), Polyoxyethylene20-cetyl ether (Brij 58), Polyoxyethylene 2-stearyl ether (Brij 72),Polyoxyethylene 10-stearyl ether (Brij 76), Polyoxyethylene 20-stearylether (Brij 78), Polyoxyethylene 2-oleyl ether (Brij 92),Polyoxyethylene 10-oleyl ether (Brij 96), Polyoxyethylene 100-stearylether (Brij 700), Polyoxyethylene 21-stearyl ether (Brij 721), and thelike); Triton X (e.g., Triton X-15, Triton X-45, Triton X-100, TritonX-114, Triton X-165, Triton X-200, Triton X-207, Triton X-305, TritonX-405, and the like); and Sorbitan (e.g., Span-20, Span-40, Span-60,Span-65, Span-80, Span-85, and the like).

Non-limiting examples of zwitterionic surfactants of interest include3-(Decyl dimethyl ammonio) propane sulfonate, 3-(Dodecyl dimethylammonio) propane sulfonate, Myristyldimethyl ammonio propane sulfonate,Hexadecyldimethyl ammonio propane sulfonate, ChemBetaine C, ChemBetaineOleyl, ChemBetaine CAS, and3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate.

Non-limiting examples of anionic surfactants of interest includeN-lauroyl sarcosine, Sodium Cocoyl Sarcosinate, Sodium MyristoylSarcosinate, Isopropyl Lauroylsarcosinate, Sodium Palmitoyl Sarcosinate,and Disodium Lauroamphodiacetate Lauroyl Sarcosinate.

In some embodiments, non-ionic surfactants are combined withzwitterionic surfactants. In certain embodiments, non-ionic surfactantsare combined with anionic surfactants. In these embodiments, the ratioof non-ionic surfactant to zwitterionic, or anionic surfactant presentin the LPM can be adjusted to achieve desired results. Non-limitingratios of interest include 25:75 non-ionic:zwitterionic surfactant,50:50 non-ionic: zwitterionic surfactant, 75:25 non-ionic: zwitterionicsurfactant, 25:75 non-ionic:anionic surfactant, 50:50 non-ionic:anionicsurfactant, and 75:25 non-ionic:anionic surfactant. A mixture ofparticular interest is a 50:50 surfactant mixture of a Brij seriessurfactant (e.g., Brij-30) and N-lauroyl sarcosine (NLS). Anothermixture of particular interest is a 50:50 surfactant mixture of a Brijseries surfactant (e.g., Brij-30) and 3-(Decyl dimethyl ammonio) propanesulfonate (DPS). As illustrated in the Examples below, thesecombinations of surfactants, when included in the LPM at a totalsurfactant concentration of 0.5-1% (w/v), provided for solubilization ofa high level of tissue constituents as assessed by total proteinconcentration, and provided for retention of bioactivity (as assessed byELISA technique).

In some specific cases, for example, the collection of live pathogens,different LPM compositions can be used to achieve desired results.Saline and tris-Hcl were used as an LPM to provide for collection of awide variety of skin-resident bacteria, and additionally, these microbesremained potent to multiply and grow ex vivo. In some embodiments, anLPM may contain an enrichment broth medium to supports growth of sampledmicrobes. Some of anaerobic bacteria are sensitive to an oxygenatmosphere. Thus, the LPM for collecting anaerobic bacteria may containa nitrogen and hydrogen atmosphere. It will be evident to the ordinarilyskilled artisan upon reading the present disclosure that LPMcompositions varying in components can be readily produced for use inspecific applications.

LPM can also include stabilizers of analytes of interest, such asprotease-inhibitors, RNase-inhibitors, and DNase-inhibitors, which canprovide for collection and at least temporary storage of analytes withminimal or no detectable degradation or loss of bioactivity. Otherexemplary liquefaction-promoting agents are described in U.S. Pat. No.5,947,921, which is incorporated herein by reference in its entirety.For example, the liquefaction-promoting agent can include surfactants,abrasive particles, and biomolecule stabilizers.

In one exemplary embodiment, the LPM is composed of a solution of 1% w/vmixture of NLS and Brij-30 in sterile PBS. In another exemplaryembodiment, the LPM is composed of a solution of 0.5% w/v mixture of DPSand Brij-30 in sterile PBS. In certain embodiments, specifically wherethe analytes are one or more proteins, the LPM contains a 1-10% v/vprotease inhibitor cocktail (e.g., catalog number: P8340, provided bySigma-Aldrich, St. Louis, Mo.). In certain embodiments, the LPM is asaline solution. In certain embodiments, the LPM is a tris-HCl solution.

LPM can also include agents defined as “sensitivity enhancers”, whichare used to stabilize liquefied tissue analytes and facilitate theiranalysis in terms of enhancing the sensitivity and specificity of thediagnostic analytical tests. As deemed necessary to achieve these goals,the sensitivity enhancers can be added into LPM prior, during or aftertissue liquefaction process, or prior or during the diagnostic analysis.For example, the sensitivity enhancer may be pre-stored in a container,and later the liquefied tissue sample may be mixed.

In typical embodiments, sensitivity enhancers are formulated ofsubstances that synergistically act with specific components of LPM (asdisclosed above) to enhance the detection sensitivity and specificity ofanalytes of interest. In an exemplary embodiment, sensitivity enhancersare formulated of substances for preventing non-specific binding ofprotein analytes present in tissue sample to various diagnostic assaysubstrates, resulting in their sensitive and specific detection. In someembodiments, sensitivity enhancers are formulated to stabilize analytesof interest by deactivating molecules such as protease, RNase and DNase.In some specific cases, sensitivity enhancers may be formulated ofsubstances that activate proteases to prevent non specific biding ofcertain analytes of interest with proteins present in the liquefiedsample. In some embodiments, sensitivity enhancers are used to adjustthe physiological state (for example, pH) of the liquefied samples tofacilitate downstream analysis of analytes of interest.

In some embodiments, sensitivity enhancer may comprise of a solvent,such as aqueous solutions (e.g., phosphate buffered saline,tris-buffered saline, etc) or organic liquids (“non-aqueous”) liquids(e.g., DMSO, ethanol, phenol and the like), which may additionallycontain but not limited to blocking reagents (e.g., Tween 20, TritonX-100, bovine serum albumin, non-fat dry milk, casein, caseinate, fishgelatin, sonicated-sperm-nucleic acids and the like), stabilizers suchas protease, protease-inhibitors, RNase-inhibitors and DNase-inhibitors,broth mediums. Depending on the type of tissue and analyte of interest,components of the sensitivity enhancer can be rationally chosen. In anexemplary embodiment, for detecting nucleic acids in liquefiedkeratinized tissue such as skin, the sensitivity enhancer comprises of100 mM NaCl, 10 mM Tris Cl (pH 8), 25 mM EDTA (pH 8), 0.5% SDS, and 0.1mg/ml protease K. Herein, Protease K may not only facilitatesliquefaction of the skin but may also stabilize nucleic acids bydecomposing DNase and RNase—present in the sample as a tissue analyte.

In some embodiments involving analyte detection by an immunoassay,sensitivity enhancer may comprise of a variety of suitable componentsincluding, but not limited to: solvent (e.g., water, a buffer solution(e.g., phosphate-buffered saline, tris-HCl, tris-buffered saline, etc),and the like), a stabilizer such as a protease inhibitor, and a blockingreagent such as Tween 20, Triton X-100, bovine serum albumin (e.g., in aconcentration range of 1-5%), non-fat dry milk (e.g., in a concentrationrange of 0.1-0.5%), casein or caseinate (e.g., in a concentration rangeof 1-5%), fish gelatin (e.g., in a concentration range of 1-5%). In anexemplary embodiment, the sensitivity enhancer for immunoassays iscomposed of a solution of 10% BSA and 0.5% Tween 20 in Tris-bufferedsaline and is mixed with the tissue sample at ratio of 1:10.

In some embodiments involving detection of nucleic acids as an analyteof interest, sensitivity enhancer may comprise of various suitablecomponents including, but not limited to: water, a buffer solution(e.g., TE, TAE, sodium citrate, etc), a chelating agents such as EDTA, astabilizer (e.g., RNase-inhibitor, DNase-inhibitor, protease, phenol,ammonium sulfate, guanidine isothiocyanate, etc), a surfactant such assodium dodecyl sulfate, and blocking reagents such assonicated-sperm-nucleic acids, Tween 20, Triton X-100, bovine serumalbumin (e.g., in a concentration range of 1-5%), non-fat dry milk(e.g., in a concentration range of 0.1-0.5%), casein or caseinate (e.g.,in a concentration range of 1-5%), fish gelatin (e.g., in aconcentration range of 1-5%). In some embodiments, where detection ofnucleic acids is desired by using polymerase-chain-reaction (PCR)technology, the LPM has to be chosen so as to avoid inclusion ofPCR-inhibitors as LPA. In exemplary embodiments, PCR-compatible LPM isTris-Hcl buffer, or EDTA buffer.

In some embodiments involving detection microbes as an analyte ofinterest, sensitivity enhancer may comprise an enrichment broth mediumso as to facilitate growth of microbes ex vivo. Some of anaerobicbacteria are sensitive to an oxygen atmosphere. Thus, the sensitivityenhancer for collecting anaerobic bacteria may contain a nitrogen andhydrogen atmosphere.

Other formulations of sensitivity enhancer for specific assay system orspecific analyte of interest will be evident to the ordinarily skilledartisan upon reading the present disclosure.

In some embodiments, the thermal properties (e.g., temperature,heat-capacity, and the like) of the LPM can be manipulated before orduring tissue liquefaction so as to reduce the adverse thermal effectsof energy exposure on tissue and/or its constituents. In one embodiment,the temperature of the LPM is maintained low enough not to inducemelting of the tissue constituents. In another exemplary embodiment, apre-cooled LPM having temperature lower than the ambient temperature(about 25° C.) can be used for ultrasound liquefaction. In anotherexemplary embodiment, the temperature of the LPM can be continuouslyreduced during energy exposure by transferring its heat to a pre-cooledliquid flowing through a heat-transfer jacket coupled to theLPM-containing reservoir.

Analytes

A variety of analytes can be detected (qualitatively or quantitatively)with the devices, methods and systems disclosed herein and, optionally,characterized to provide an analyte profile of the tissue in question.Non-limiting examples include: structural and signaling proteins (e.g.,keratins (e.g., basic keratins, acidic keratins), β-actin, interleukins,chemokines, growth factors, colony-stimulating factors, interferons,antibodies (IgE, IgG, IgA, IgD, IgM), cancer biomarkers (e.g., CEA, andthe like), heat shock proteins (e.g., Hsp-60, Hsp-70, Hsp-90, etc.), andthe like, lipids (e.g., cholesterol), ceramides (e.g., ceramides 1-6),fatty acids, triglycerides, paraffin hydrocarbons, squalene, cholesterylesters, cholesteryl diesters, free fatty acids, lanosterol, cholesterol,polar lipids (e.g., glucosyl-derivatives and phospholipids), and thelike, nucleic acids (e.g., RNA and DNA), small molecules (e.g., freeamino acids, lactate, exogenously delivered drug molecules,environmental contaminants, warfare agents, and the like) andmicroorganisms (e.g. bacteria, fungi, viruses and the like). Theseanalytes are found within the tissue itself, and may not be solelypresent in the interstitial fluid around the tissue. The analyte may beother than a marker associated with interstitial fluid, such as a tumormarker. Thus, the devices, methods and systems disclosed herein can beadapted to detect tumor markers that are present in tissue structures,but which may or may not also be present in interstitial fluid.

In a particular embodiment, antibodies against allergens and cytokinesare liquefied (are these liquefied or is the tissue liquefied to producethese soluble analytes) and characterized to provide an allergy profilefor the tissue and the subject in question. Specific types of antibodiesinclude but are not limited to IgE and IgG antibodies. Specific types ofcytokines include but are not limited to IL4, IL5, IL10, IL-12, IL13,IL-16, GM-CSF, RANTES, MCP-4, CTACK/CCL27, IFN-g, TNFa, CD23, CD-40,Eotaxin-2, and TARC.

The analytes can be analyzed in many ways, which can be readily selectedby the ordinarily skilled artisan in accordance with the analyte to beevaluated. A reservoir or collecting container can be applied to thesite for collection of sample, which is then measured using analyticaltechniques. Application of energy can be optimized to maximize analyterecovery. It may be desirable for certain applications to maintain therelative levels of the analyte to other components of the sample.Exemplary assay methods include but are not limited to gelelectrophoresis, agar plating, enzymatic testing, antibody-based tests(e.g., western blot tests, Enzyme-Linked Immuno Sorbent Assay (ELISA),lateral flow assays, and the like), thin layer chromatography, HPLC,mass spectrometry, radiation-based tests, DNA/RNA electrophoresis,(UV/Vis) spectrophotometry, flow assays, and the like.

A quantitative measurement of the presence of tissue constituents in theliquefied tissue sample can assess the extent of tissue liquefaction.Such an internal calibration can be accomplished by measuring one ormore optical properties of the liquefied tissue sample such asabsorbance, transmittance, scattering, or fluorescence emission uponbeing irradiated by a source emitting electromagnetic waves. Additionalsample parameters such as gravimetric-weight, total protein content, pH,and electrical conductance can be used for calibrating the extent ofliquefaction. Further, measurement of tissue properties such asthickness, rate of water loss, and electrical conductivity can be used.Direct measurement of the concentration of one or more sampled analytessuch as β-actin, β-tubulin, GAPDH (glyceraldehyde 3-phosphatedehydrogenase), LDH (lactate dehydrogenase), or any otherabundantly-present biomolecule whose concentration is expected to remainconstant in the tissue, can be used for calibrating the extent of tissueliquefaction. Analytes could also be quantified using immunologicalbased assay (i.e. radioimmuno; Eliza; FACs).

Tissue Cells and Microorganisms

In addition to the analytes described above, whole cells of tissue underanalysis, as well as a variety of microorganisms, can be detected intissues of interest using the devices, methods and systems disclosedherein. Tissue cells and most microorganisms are much larger than theanalytes described above, and their extraction from a tissue of interestcan be accomplished using various embodiments of the current invention.Pathogenic and nonpathogenic bacteria, virus, protozoa, and fungi playwell-known roles in various infectious diseases, and their detection canfacilitate a diagnosis of a disease caused by the microorganism (e.g.,tuberculosis, herpes, malaria, ringworm, etc.). The disease stateexhibits either the presence of a novel microorganisms or an alterationin the proportion of resident microorganisms. When a subject issuspected of having an infection with such a microorganism, the devices,methods and systems disclosed herein can be used to quantify or detectthe presence or absence of a microorganism, and facilitate diagnosis ofthe condition.

Non-pathogenic microorganisms are normally present in healthy tissues(“normal flora”), and can play a role in many bodily functions andmaintenance of health of a subject. Detection of these normal floramicroorganisms (e.g., bacteria) in a tissue of interest can also beaccomplished with the current method and device. A subject's tissue canbe sampled and analyzed using the devices, methods and systems disclosedherein to examine the various microorganisms that are naturally present.When a subject is suspected to have an abnormal condition, tissue of thesubject can be sampled according to the devices, methods and systemsdisclosed herein to detect the presence or absence of a change in aprofile of non-pathogenic microorganisms relative to that of a normal,healthy subject. A change in this microorganism profile can facilitatediagnosis of a condition of interest in the subject.

In some embodiments, tissues can be liquefied to recover their cells ormicroorganisms residing therein. Application of the present devices,methods and systems using energy provide for collection of bacteria fromskin of a subject into a collection medium which may optionally containan LPA. For example, application of ultrasound energy to the tissue ofinterest using tris-Hcl or PBS is sufficient to collect bacterialmicroflora. In general, use of a device utilizing this method involvesapplication of a sufficient level of ultrasound energy so as to dislodgemicroorganisms from the tissue and enter the collection medium, which isthen collected for subsequent analysis, which may include culturing themedium to determine whether certain microorganisms are present, directlyassaying the medium (e.g., using ELISA techniques, e.g., involvingmicroorganism-specific antibodies, e.g. involving a latex agglutinationtest, e.g., using a nucleic-acid-based diagnostic assay including thepolymerase chain reaction hybridization, DNA sequencing method), or acombination of these approaches. Detection of microorganisms in themedium facilitates diagnosis of a condition of interest. Furthermore, ahigh yield collection of microorganisms could shorten or eliminate aprocess to amplify the number of nucleic acids for diagnostics.

The invention described herein can also be used to collect cells fromthe tissue. Application of energy with an appropriate LPM that liquefiestissues without disrupting cell membranes can be used to harvest wholecells, including viable whole cells from tissues. LPM in this case maycomprise chemicals including but not limited to ion chelating agentssuch as EDTA or enzymes such as trypsin to dislodge the cells.Similarly, with changes in parameters of energy and/or LPM as discussabove, the devices, methods and systems of the present disclosure can beused to collect nuclei or other cellular organelles.

Tissue of Interest

A variety of tissues are well suited to the devices, methods and systemsdisclosed herein. These tissues include but are not limited to skin,mucosal membranes (nasal, gut, colon, buccal, vagina etc.) or mucus,breast, prostate, eye, intestine, bladder, stomach, esophagus, nail,testicles, hair, lung, brain, pancreas, liver, heart, bone, or aortawall. In one embodiment, the tissue is skin, which can be skin of theface, arms, hands, legs, back, or any other location. While skin andmucosal surfaces are highly accessible for performing liquefaction,liquefaction devices, methods and systems described in this disclosurecan be designed to readily adapt to various internal tissues listedabove. Exemplary devices specific to internal tissues that can find usein the methods disclosed herein include those disclosed in U.S. Pat. No.5,704,361, U.S. Pat. No. 5,713,363, and U.S. Pat. No. 5,895,397, each ofwhich are incorporated herein by reference in their entirety.

In some embodiments, the tissue of interest is other than a tumor or atissue suspected of being a tumor. Where the devices, methods andsystems disclosed herein are applied to detection of a microorganism,the tissue of interest is one suspected of containing a microorganism(e.g., a tissue suspected of having an infection, particularly a deeptissue infection, e.g., infection of the dermal and/or subdermal layersof the skin, including such layers of mucosal membranes).

Method of Use

The methods disclosed herein can be used for a broad range of tissueevaluations, including assessment of the presence or absence of ananalyte(s) of interest to facilitate diagnosis of a condition ofinterest. In some embodiments, the methods find use where, for example,the patient presents with clinical signs and symptoms suggestive of oneor more conditions, where the methods disclosed herein can facilitate adifferential diagnosis.

In certain embodiments, the current invention provides methods thatinvolve comparing a test analyte profile generated from a patient sampleto a reference analyte profile. A “reference analyte profile” or“analyte profile for a reference tissue” generally refers to qualitativeor quantitative levels of a selected analyte or set of 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more analytes, which are characteristic of a condition ofinterest. Exemplary conditions of interest for which a reference analyteprofile may be provided include, but are not limited to, normalreference analyte profile (e.g., healthy tissue (i.e., absence ofdisease), general tissue health, acceptable or tolerated levels of ananalyte (e.g., a drug, environmental contaminant, etc.), diseasereference analyte profile (e.g., an analyte profile characteristic ofthe presence of, for example, microbial infection (e.g., bacterial,viral, fungal, or other microbial infection), localized diseases intissues (e.g., dermatitis, psoriasis, cancers (prostate, breast, lung,etc.), urticaria, etc.), systemic diseases manifested in tissues (e.g.,allergies, diabetes, Alzheimer's disease, cardio-vascular diseases, andthe like); etc.), environmental contaminant reference analyte profile(e.g., an analyte profile characteristic of the presence of unacceptablyhigh levels of an environmental contaminant (e.g., warfare agent,pollens, particulates, pesticides, etc.), drug reference analyte profile(e.g.. an analyte profile characteristic of therapeutic levels of adrug, drug-of-abuse (e.g., to facilitate assessment of drug-of-abuse),etc.); and the like. Reference analyte profiles may include analytesthat are members of one or more classes of analytes (e.g., proteins(e.g., antibodies, cancer biomarkers, cytokines,cytoskeletal/cytoplasmic/extra-cellular proteins, and the like), nucleicacids (DNA, RNA), lipids (which include ceramides, cholesterol,phospholipids, etc.), biologically-derived small molecules, drugs (e.g.,therapeutic drugs, drugs-of-abuse), environmental contaminants, warfareagents, etc.) or members of a subclass of analytes (e.g., antibodies,phospholipids). Reference analyte profiles of a given condition ofinterest may be previously known in the art or may be derived from thetissue using the methods described in this invention. Reference analyteprofiles can be stored in electronic form (e.g., in a database) toprovide for ready comparison to a test analyte profile to facilitateanalysis and diagnosis.

A “test analyte profile” or “analyte profile for a tissue of interest”refers to qualitative or quantitative levels of a selected analyte orset of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more analytes, to facilitatediagnosis or prognosis of a condition of interest. A test analyteprofile may include analytes that are members of one or more classes ofanalytes (e.g., proteins, nucleic acids, lipids, biologically-derivedsmall molecules, drugs (e.g., proteins (e.g., antibodies, cancerbiomarkers, cytokines, cytoskeletal/cytoplasmic/extra-cellular proteins,and the like), nucleic acids (DNA, RNA), lipids (which includeceramides, cholesterol, phospholipids, etc.), biologically-derived smallmolecules, drugs (e.g., therapeutic drugs, drugs of abuse),environmental contaminants, warfare agents, etc.) or members of asubclass of analytes (e.g., antibodies, phospholipids). In general, theanalytes selected for analysis to generate a test analyte profile areselected according to analytes of a desired reference analyte profile.Comparison of a test analyte profile to an appropriate reference analyteprofile facilitates determining the presence or absence of the conditionor state of interest, e.g., by assessing whether there is a substantial“match” between a test analyte profile and a reference analyte profile.

Methods for generating reference and test analyte profiles of a selectedanalyte or set of analytes can be accomplished using methods availablein the art, and will be selected according to the analyte(s) to beassessed.

The current methods can be used for a broad range of tissue evaluations.Energy-assisted tissue liquefaction can provide a quantitativeevaluation and profile of normal tissue. Comparison of the normal tissueprofile with a profile of tissue under investigation can facilitatediagnosis of changes in tissue microenvironment (e.g. up/down-regulationof several proteins, lipids, nucleic acids, small molecules, drugs, etc)which can indicate various diseased conditions such as allergies,cardio-vascular disease, dermatitis, etc. The methods can also be usedas a tool for monitoring tissue recovery and evaluating therapeuticefficacy of various treatments (as in monitoring of therapy, which canbe combined with modification of therapy as desired or needed). Theanalyte profiling methods can also provide tools for the personal-careindustry for evaluation of topical formulations (e.g., as in cosmetics).This methodology can be utilized for determining pharmacologicparameters by liquefying tissues and detecting the drug moleculestherein. In a similar manner, rapid and routine testing of chemicals,bio-hazardous contaminants, and drugs-of-abuse can also bequantitatively accomplished. The methods can also be used for sensitivedetection and diagnosis of pathogenic microflora.

In certain embodiments, the current methods provide a profile of normaltissue, wherein normal tissue is defined by the absence of the abnormaltissue condition of interest. Energy is applied to the normal tissue,e.g., by ultrasound exposure or abrasion, in the presence of aliquefaction-promoting agent. Various tests are performed upon theliquefied tissue sample to isolate and identify the analytes present inthe tissue.

In certain embodiments, the methods can be applied to facilitatediagnosis of various tissue diseases which are characterized by aquantitative evaluation of a change in the tissue microenvironment. Thisevaluation is performed by comparing an analyte profile of a referencetissue (e.g., a reference analyte profile, which may be stored in adatabase) with the analyte profile of the tissue of interest (i.e., thetest analyte profile). The quantitative presence or absence of a certainanalyte or set of analytes present in a tissue under investigation, whencompared to the quantitative presence or absence of the same analytes ina reference tissue will indicate the presence or absence of a particulardisease, and thus facilitate diagnosis of the condition. The referenceanalyte profile can be one characteristic of tissue which is known tonot be affected with the disease in question, or can be a referenceanalyte profile characteristic of the disease in question for the tissuein question.

In one embodiment, the tissue under investigation is skin and/or mucosalmembranes, and the quantitative test analyte profile is compared to areference analyte profile to determine the presence or absence of adisease such as allergy, urticaria, microbial infection, auto-immunedisease, cardiovascular disease, or cancer.

In certain embodiments, this method can be used to monitor tissuerecovery. This monitoring is performed by comparing an analyte profileof reference tissue with the analyte profile of tissue underinvestigation. The quantitative presence or absence of a certain analyteor composition of analytes present in a tissue under investigation, whencompared to the quantitative presence or absence of the same analytes ina reference tissue can indicate whether or not the tissue is returningto its healthy state. The reference tissue is usually tissue that is ina healthy state.

In certain embodiments, the current methods can be used to evaluate thetherapeutic effect of various treatments, including bioavailability oftherapeutics in tissues of interest. The analyte in the liquefied tissuesample can be quantified to indicate how much of the analyte is presentin the tissue. The quantitative presence or absence of a certain analyteor composition of analytes present in a tissue under investigation, whencompared to the quantitative presence or absence of the same analytes ina reference tissue, can indicate whether or not the dosed therapeuticagent is staying in the specific tissue or body long enough to achieveits desired effect. The reference tissue is usually tissue that is in ahealthy state.

In certain embodiments, the methods disclosed herein can be used toevaluate therapeutic formulations on a tissue such as skin,specifically, whether component(s) of a formulation (e.g., lotions,creams, salves, and the like) are being absorbed by the tissue, and ifthe amount delivered is therapeutically effective. In certainembodiments, the methods disclosed herein can include a closed loopsystem, in which the same system can apply the therapeutic formulation,liquefy the analytes, analyze the analyte profile, and adjust thedelivery of the formulation accordingly. The reference tissue in thiscase would be healthy tissue, or tissue at various levels of recoveryfrom the condition that the therapeutic formulation was treating.

In certain embodiments, the current methods can be used to determine theanalyte profile for use in determining pharmacological parameters orefficacy of pharmaceutical agents. The presence or absence of certainanalytes (e.g., immune system responders, cytokines) can be used tocorrelate certain dosages of pharmaceutical agents to biologicalparameters, including but not limited to bioavailability, AUC,clearance, and half life.

In certain embodiments, the methods disclosed herein can be used todetect the presence or absence of certain chemicals, including but notlimited to bio-hazardous contaminants, warfare agents, illicit drugs,known pharmaceutical agents, and the like. Such methods find use in, forexample, law enforcement, regulation of doping in competitive sports,evaluation of exposure and/or risk of disease as a result of exposure totoxins or contaminants, and the like.

In certain embodiments, the current methods can be used for detecting ordiagnosing pathogenic microbes (e.g., bacteria, fungi, viruses, and thelike). Current methodologies for microbial diagnostics in tissues, suchas replica plating, swabbing, and washing, are unattractive due to largevariability and low dispersion of extracts, which leads to decreasedsensitivity and high protocol-dependency. Various tests can be performedupon the liquefied tissue sample to isolate and identify the microbialanalytes present in the tissue. In certain embodiments, these testsinclude plating on agar plates.

Drug Delivery

The present invention provides a method and device involvingliquefaction of a tissue so as to control and enhance the flux of drugsinto or through the tissue. The method includes the steps of 1) applyingenergy and a liquefaction promoting medium to a tissue where transportis desired of a subject; and 2) delivering one or more drugs into orthrough the tissue to be liquefied continuously or repeatedly. Themethod may further include reliquefy the tissue over the period of timeduring which transport occurs. The method comprising liquefying a tissuecan perturb the barrier properties of a tissue or biological surface,leading to reducing the resistance to the drug's passage. The advantageof the present invention is that the rate and efficiency of transfer isboth improved and controlled. Drugs which would simply not pass throughthe biological surfaces, or pass at a rate which is inadequate orvariable over time, are forced into the biological surfaces when energyin combination of a LPM is applied. By controlling the mode, intensityand time of energy application and formulation of a LPM, the rate oftransfer is controlled.

The transport of drugs can be modulated or enhanced by the simultaneousor subsequent application of a secondary driving force such as chemicalpermeability or transport enhancers, convection, osmotic pressuregradient, concentration gradient, iontophoresis, electroporation,magnetic field, ultrasound, or mechanical pressure.

Enhancement of the disclosed method was demonstrated by the followingnon-limiting example employing ³H-labelled Acyclovir and Inulin. Therequired type, length of time, and intensity of energy and formulationof a LPM are dependent on a number of factors including the type oftissues and the property of drugs, which varies from species to species,with age, injury or disease, and by location on the body.

Drug to be Administered

Drugs to be administered include a variety of bioactive agents, but arepreferably proteins or peptides. Specific examples include insulin,erythropoietin, and interferon. Other substances, including nucleic acidmolecules such as antisense, siRNA and genes encoding therapeuticproteins, synthetic organic and inorganic molecules includinganti-inflammatories, antivirals, antifungals, antibiotics, localanesthetics, and saccharides, can also be administered. The drug willtypically be administered in an appropriate pharmaceutically acceptablecarrier having an absorption coefficient similar to water, such as anaqueous gel. Alternatively, a patch can be used as a carrier. Drug canbe administered in a gel, ointment, lotion, or suspension.

In one embodiment, the drug is in the form of or encapsulated in adelivery device such as liposome, lipid vesicle, emulsion or polymericnanoparticles, microparticle, microcapsule, or microsphere (referred tocollectively as microparticles unless otherwise stated). These can beformed of polymers such as polyhydroxy acids, polyorthoesters,polyanhydrides, and polyphosphazenes, or natural polymers such ascollagen, polyamino acids, albumin and other proteins, alginate andother polysaccharides, and combinations thereof. The microparticles canbe coated or formed of materials enhancing penetration, such aslipophilic materials or hydrophilic molecules, for example, polyalkyleneoxide polymers, and conjugates, such as polyethylene glycol.

Administration of Drug

The drugs are preferably administered, using the liquefaction devicesmentioned, to the tissues at a site selected based on convenience to thepatient as well as to achieve desired treatment results. A variety oftissues including biological surfaces are well suited to the currentmethod. These tissues include but are not limited to skin, mucosalmembranes (nasal, gut, colon, buccal, intestine, vagina, etc.). In oneembodiment, the method of the current invention is preferablyadministered to the skin of the face, arms, hands, legs, back, or anyother location. While skin is highly accessible for performingliquefaction, the devices described in this disclosure can be designedto readily adapt to various internal membranes listed above.

In some embodiment, the tissue to be administered is a diseased tissuesuch as infectious organs, tissues that is inflamed, and solid tumors.In a certain embodiment, the present invention comprises using theliquefying devices on the healthy tissues in the vicinity of and/or thediseased tissue, and delivering drugs across the healthy tissues and/orinto the site of the diseases. Steroids such as corticosteroids and manyof chemotherapeutic agents including estramustine phosphate, paclitaxel,and vinblastine have potentially severe side effects. Hence, if givensystemically, they are likely to cause undesirable side effects. Thisproblem is overcome by delivering these drugs locally to the diseasetissues. Other indications include delivery of drugs into abnormal skinsuch as psoriasis, atopic dermatitis, and scars.

In some embodiments, the current invention is used to enhance thepassage of a compound such as a large molecular weight or polar moleculethrough the tissue such as skin, mucosal membranes (nasal, gut, colon,intestine, buccal, vagina etc.). Greater control and drug utilizationare achieved by increasing the rate and directional control of theapplied drug. The percentage of drug which quickly enters thebloodstream is increased accordingly and undesirable side effects areavoided. Drugs through the tissues stated above are infused into thebloodstream at an optimal rate.

Liquefaction Promoting Medium (LPM) for Drug Delivery

A LPM is also an important component for drug delivery. The design ofthe LPM for drug delivery is overlapping somewhat to that of the LPM forsample collection. The LPM can be designed to serve one or more of thefollowing five purposes: a) it couples energy to a tissue, b) itfacilitates liquefaction of the tissue, c) it storages drugs to bedelivered into the tissue, d) it increases the solubility of the drugs,and e) it inhibits degradation of the drugs such that their biologicalor chemical activity is retained.

The LPM may also contain a drug prior or during tissue liquefactionprocess. In an alternate embodiment, application of energy and the LPMwhich excludes a drug can be used for liquefying a tissue, andsubsequently a drug in an appropriate carrier such as a patch can beapplied on a site of the tissue to be liquefied.

Kits

The present disclosure also encompasses kits for practicing the currentmethods. The subject kits can include, for example, the entire energyapplication device and a liquefaction-promoting agent to liquefy tissuesof interest, reagents for conducting assays to detect and analyze(qualitatively or quantitatively) the presence or absence of tissueanalytes in the liquefied tissue sample generated through methodsdisclosed herein. The various components of the kit may be present inseparate containers, or certain compatible components may bepre-combined into a single container, as desired.

In addition to the above-mentioned components, the kits typicallyfurther include instructions for using the components of the kit topractice the methods. The instructions for practicing the subjectmethods are generally recorded on a suitable recording medium. Forexample, the instructions may be printed on a substrate, such as paperor plastic, etc. As such, the instructions may be present in the kits asa package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging orsub-packaging) etc. In other embodiments, the instructions are presentas an electronic storage data file present on a suitable computerreadable storage medium, e.g. CD-ROM, diskette, etc. In yet otherembodiments, the actual instructions are not present in the kit, butmeans for obtaining the instructions from a remote source, e.g. via theinternet, are provided. An example of this embodiment is a kit thatincludes a web address where the instructions can be viewed and/or fromwhich the instructions can be downloaded. As with the instructions, thismeans for obtaining the instructions is recorded on a suitablesubstrate.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep, or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

Example 1 Sampling of Skin by an Abrasive Energy-Based Device

Referring to FIGS. 7 a through 7 e, an abrasive energy-based tissueliquefaction device for sampling of skin tissue is described. Device isassembled from three components—751 (assembly of device housing 701containing motor 704 and electrical conductivity components 705 and706); 752 (disposable assembly of LPM cartridge 708, collectioncontainer 707 and needle 709); and 753 (disposable assembly of LPMhousing 712, abrasive pad 711 and shaft 710) (FIG. 7 a). The assembleddevice is placed against a pre-identified region of interest on skin713, such that abrasive pad 711 is facing skin 713 (FIG. 7 b). Slidingplunger 702 located on top of the device is pushed towards skin, whichpushes needle 709 into LPM cartridge 708, breaking its sterile seal andtransfers LPM into housing 712. Sliding plunger 702 also energizes motor704 through battery pack 703, setting the shaft 710 and abrasive pad 711in rotary motion against skin tissue 713. As skin tissue is liquefied,tissue components are dissolved in LPM contained in housing 712.Electrical conductivity of skin tissue 713 is also simultaneouslymeasured using sliding contact 705 fastened to shaft 710 as measurementelectrode and reference electrode 706. Once the safe energy exposurelimit is reached as determined by threshold electrical conductivity,motor 704 stops. Sliding plunger 702 is further pushed towards skin suchthat needle 709 punctures a pre-vacuumized sample container 707, whichaspirates the sample from housing 712 in it (FIG. 7 d). The device isremoved form skin and disassembled. The device component 752 is furtherdissembled and sample container 707 is processed for detection ofanalytes.

Example 2 Sampling of Skin by a Microneedle-Based Device

Referring to FIGS. 8 a through 8 d, a microneedle-based tissueliquefaction device for sampling of skin tissue is described. The deviceis placed against a pre-identified region of interest on skin 807, suchthat microneedle bearing patch 805 is facing skin 807 (FIG. 8 a).Sliding plunger 801 located on top of the device is pushed towards skin807 such that LPM soaked sponge 804 is squeezed and releases LPM intohousing 803 (FIG. 8 b). Consequently, microneedles in patch 805 andhousing 803 at the skin interface are filled with LPM. To initiateliquefaction process, sliding plunger 801 is further pushed into skintissue 807 leading to insertion of microneedles 805 into skin tissue 807(FIG. 8 c). As skin tissue is liquefied, tissue components are dissolvedin LPM contained in housing 803. Upon completion of skin liquefaction,pre-vacuumized sample container 802 is pushed towards skin tissue 807such that needle 806 punctures sample container 802 resulting inaspiration of sample from housing 803 in it (FIG. 8 d). The device isremoved from skin and disassembled. Sample container 802 is retrievedfor analyte analysis and the rest of the device components are disposedoff.

Example 3 Reservoir Housing for Capturing Tissue Analytes

Referring to FIG. 9 (Panels a-d) a design for a reservoir housing tocapture tissue analytes from liquefied tissue samples is described. Thereservoir housing (901) is intended to be used with energy-applicationdevices described herein, as a container to collect the liquefied tissuesample. The housing is coated with capture substrates (902) whichselectively bind to tissue analytes (903) present in the sample. Uponsufficient incubation of the tissue sample, the sample is discardedwhile the analytes (903) are held in the housing. The analytes areeluted by an elution buffer in the housing for subsequent capture of theanalytes as a separate sample (904). Alternatively, the housing can beintegrated in an analytical tool for analyzing the bound analytes (903).

Example 4 Surfactant Formulations for Enhanced Tissue Solubilization andProtein Functionality Retention

Unique surfactant formulations were identified that make up theliquefaction promoting medium (LPM) according to the definitiondisclosed in this text. A library of 153 binary surfactant formulationswas created using 19 surfactants belonging to four distinct categories:(i) anionic surfactants (sodium lauryl sulfate (SLS), sodium laurethsulfate (SLA), sodium tridecyl phosphate (TDP), sodium deoxycholate(SDC), sodium decanoyl sarcosinate (NDS), sodium lauroyl sarcosinate(NLS), sodium palmitoyl sarcosinate (NPS)); (ii) cationic surfactants(octyl trimethyl ammonium chloride (OTAB), dodecyl trimethyl ammoniumchloride (DDTAB), tetradecyl trimethyl ammonium chloride (TTAB)); (iii)zwitterionic surfactants (3-[(3-Cholamidopropyl) dimethylammonio]1-propane sulfonate (CHAPS), 3-(Decyl dimethyl ammonio) propanesulfonate (DPS), 3-(Dodecyl dimethyl ammonio) propane sulfonate (DDPS));(iv) nonionic surfactants (Polyethylene glycol dodecyl ether (B30),Polyoxyethylene 23-lauryl ether (B35), Polyoxyethylene 10-cetyl ether(B56), Polyoxyethylene 2-stearyl ether (B72), Polyethylene glycol oleylether (B93), Nonylphenol polyethylene glycol ether (NP9)). Only ahandful of surfactants from these categories (for example, nonionicsurfactants) have been traditionally utilized for extracting functionaltissue proteome. Additionally, these surfactants are highly limited intheir ability to efficiently solubilize tissue constituents. As such,across all surfactant types, extraction potential and bioactivitypreservation of tissue constituents are largely considered as mutuallyconflicting properties. By combining nonionic surfactants with othertypes of surfactants that have been previously described for their highsolubilization ability (anionic, cationic and zwitterionic surfactants),we show the discovery of new families of surfactant formulations thatsimultaneously possess superior solubilization as well as non-denaturingcapabilities.

The surfactant library was first screened for identifying non-denaturingsurfactant formulations that retain protein bioactivity in extracts, andsubsequently ranked for the ability of formulations to solubilize tissueproteins. FIG. 10 a shows the potency of 153 surfactant formulations topreserve the specific functionality of a model protein—IgE antibody.Specifically, binding ability of IgE antibody with ovalbumin was tested.The x-axis in this figure represents the formulation index unique toeach binary formulation. The y-axis represents % IgE bioactivityretention, defined as the fractional IgE binding activity in surfactantformulation compared with IgE binding activity when surfactants in puresolvent (phosphate buffered saline, PBS). The formulations spanned awide range of denaturing potentials. Surprisingly, an increasing numberof denaturing surfactants upon combination with gentler nonionicsurfactant yielded a high synergistic gain in IgE functionalityretention. Non-denaturing potential, averaged over all binary surfactantformulations was found to be significantly higher than their constituentsingle surfactant formulations (p<0.006; two-tailed heteroscedasticstudent's t-test); further demonstrating unique synergic interactions.

Surfactant formulations exhibiting high bioactivity retention (≧90%)were further screened for their ability to extract tissue proteins inconjunction with a brief sonication treatment. Porcine skin was used asa model tissue for these studies. While a majority of formulationsrevealed an extraction potential close to 0.1 mg protein per cm² of skintissue, only a couple of formulations achieved protein extractionexceeding 0.3 mg/cm² (FIG. 10 b).

The leading candidates screened from the surfactant library generallyresulted in formulations that were exceptionally non-denaturing, yetmore effective in solubilizing tissues than some of the most widely usedextraction surfactants reported in the literature. FIG. 10 c comparesthe leading surfactant formulation—0.5% (w/v) DPS-B30, with 1% (w/v) SDSfor skin sampling. Despite a moderate extraction ability (0.16±0.07mg/cm²), SDS is highly denaturing which results in a low yield offunctional protein recovery (product of fractional bioactivity retainedand total extracted protein). In contrast, 0.5% (w/v) DPS-B30formulation not only extracts more skin proteins (0.48±0.12 mg/cm²) butalso preserves protein activity, amounting to an excess of 100-foldenhancement in expected functional protein recovery over SDS. Similarly,more than 10-folds of protein recovery were accomplished over commonlyused non-denaturing surfactant 1% (w/v) Triton X-100 and PBS.

Example 5 Bioactivity Retention Under Stress

We show that unique surfactant formulations, or LPMs (as indentified bymethod described in Example 1) additionally protect a variety ofanalytes under stress. We specifically show denaturing effects ofmechanical energy such as ultrasound exposure (a commonly knowndenaturant to biomolecules), can be neutralized with the use of uniquesurfactant formulations.

In separate experiments, a globular protein (IgE) and two representativeenzymes—lactate dehydrogenase (LDH) and beta-galactosidase (β-Gal) weredissolved in 0.5% (w/v) DPS-B30 surfactant formulation and sonicated todetermine retention of protein bioactivity over time. Proteins dissolvedin saline (PBS) were prepared as comparative controls. A progressivelysharp decrease in functionality was observed for IgE dissolved in PBS;however, 0.5% (w/v) DPS-B30 formulation, surprisingly, extendedprotection to IgE proteins towards ultrasonic denaturing stress (FIG. 11a). Irrespective of ultrasound treatment, IgE dissolved in SDS showedcomplete state of denaturation. Similar trends were observed on extendedpreservation of enzymatic activities for LDH and β-Gal prepared in 0.5%(w/v) DPS-B30 formulation (FIG. 11 b). Protein preparation in PBSresulted in significant loss of bioactivity (p<0.006; two-tailedheteroscedastic student's t-test), amounting to a fractional bioactivityof 16.7% (IgE), 70.8% (LDH) and 68.7% (β-Gal) after 3 minutes ofsonication.

Example 6 Tissue Sampling And Molecular Diagnostics

The ability of ultrasonic exposure in the presence of LPM (salinesolution of 0.5% (w/v) DPS-B30) to sample a variety of functionaldisease biomarkers from tissues was demonstrated.

Sampling of allergy-specific IgE antibodies from the skin of miceallergic to egg was demonstrated. Six to eight weeks old female BALB/CJmice were purchased from Charles River Labs (Wilmington, Mass.) andmaintained under pathogen-free conditions. Allergic reaction was inducedin mice by an epicutaneous exposure protocol. After anesthesia with1.25-4% isofluorane in oxygen, the skin on the back of the mice wasshaved and then tape stripped 10-times (Scotch Magic tape, 3M HealthCare, St Paul, Minn.) to introduce a standardized skin injury. A gauzepatch (1 cm×1 cm) soaked with 100 μL of 0.1% OVA was placed on the backskin and secured with a breathable elastic cloth-based adhesive tape.The patches were kept affixed for 1 week. The whole experiment compriseda total of three 1-week exposures with a 2-week interval between eachexposure week. Sampling was performed by gluing a custom made flangedchamber (skin exposure area of 1.33 cm²) to the shaven skin area with aminimal amount of cyanoacrylate-based adhesive. The chamber was filledwith 1.8 ml of 0.5% (w/v) DPS-B30 surfactant formulation and 20 kHzultrasound was applied at 50% duty cycle, 2.4 W/cm² for 5 minutes. Skinbiopsies of ultrasound treated or untreated eczema skin sites wereobtained, and skin homogenate samples were prepared as positivecontrols. FIG. 12 a shows that ultrasound-assisted sampling successfullysampled significantly more amount of allergy-specific IgE antibodiesfrom allergic mice skin as compared to healthy mice. Expectedly, nodifference was seen in the amount of IgG antibodies in the samples fromallergic and healthy mice skins.

Sampling of cholesterol from mouse skin was also demonstrated. Withsimilar procedures as described in above paragraph, skin samples withthe ultrasound procedure were collected. Skin homogenates were preparedas positive controls from biopsies collected from untreated skin. Skincholesterol is an important biomarker for diagnosing cardiovasculardisease [1]. FIG. 12 b shows that ultrasound-assisted samplingsuccessfully samples cholesterol from skin and the amount sampled iscomparable to the cholesterol present in skin homogenate.

Lastly, sampling of bacterial genome from porcine skin was demonstrated.Tissues, particularly skin and mucosal membranes are colonized by adiverse set of microorganisms including bacteria, fungi and viruses[2-5]. Accurate diagnosis of bacterial infection leads to appropriatepatient management, providing information on prognosis and allowing theuse of a narrow-spectrum antibiotics [6-8]. Thus, definitivemicroorganism detection is essential for diagnosis for treatment ofinfection and trace-back of disease outbreaks associated with microbialinfections. Accurately obtaining samples which represent microorganismson skin, however, is a major challenge [2]. The most practical method ofcollection would be swabbing because it is simple, quick and noninvasive[3, 9]. However, swabbing has several limitations including poorrecoveries of the microorganisms and lack of a standardized protocol,which suggests it either does not accurately represent themicroorganisms on the skin or provide quantitative data.Ultrasound-assisted sampling can effectively address these limitations.In particular, excised porcine skin was sampled by swabbing with acotton ball soaked in saline (PBS), and by ultrasound-assisted samplingwith 0.5% (w/v) DPM-Brij30 as LPM in separate experiments. The bacterialgenome was purified from each sample by standard phenol-chloroformextraction method. Briefly, samples were first incubated in a solutionconsisting of 20 mM Tris at pH 8.0 (BP154-1, Fisher Scientific), 2 mMEDTA(BP120-500, Fisher Scientific), 1.2% Triton X-100 (BP151-100, FisherScientific), and 20 mg/ml lysozyme (62970-1G-F, Sigma-Aldrich) for 30min at 37° C. [9]. Subsequently, samples were incubated for 3 hours at37° C. in a solution consisting of 0.1 mg/ml Proteinase K (P2308-25MG,Sigma-Aldrich), 0.5% (w/v) sodium lauryl sulfate (S529, FisherScientific), and 100 mM sodium chloride (BP358-1, Fisher Scientific).Genomic DNA was then extracted with an equal volume of phenol (P4557,Sigma-Aldrich), followed by extraction with phenol/chloroform/isoamylalcohol, 25:24:1 (P2069, Sigma-Aldrich). The DNA was precipitated byincubation with ethanol and centrifugation for 20 min. The DNA pelletswere washed twice with 70% ethanol, allowed to dry, and resuspended in80 μl of tris buffer. The amount of bacteria sampled by each methodologywas evaluated by determining the presence of the conserved 16S bacterialgene in each sample using quantitative-polymerase-chain-reaction (qPCR).FIG. 12 c shows that ultrasound-assisted sampling sampled at least7-fold higher amount of bacterial genome from skin than the conventionalcotton swabbing procedure.

Example 7 Buffer Design of LPMs Compatible With Nucleic-Acid-Based Tests

To ensure compatibility of the liquefied tissue samples with subsequentanalysis, the components of LPMs have to be carefully chosen.Compatibility of several LPM components with nucleic-acid-basedanalytical technique was tested. Specifically, the compatibility of LPMcomponents with qPCR—the most common gene-based test, was evaluated bymeasuring the test's ability to amplify plasmid DNA added in differentLPMs.

Ten million copies of Luciferase plasmid (E1741, Promega Corp.) werespiked in 10 μl of different solutions: (i) water, (ii) 0.91% (w/v)sodium chloride (BP358-1, Fisher Scientific) in water, (iii) PBS (P4417,Sigma-Aldrich), (iv) 10 mM Tris-HCl, pH 7.9 (BP154-1, FisherScientific), (v) 0.075 M sodium phosphate buffer, pH 7.9, derived fromsodium phosphate monobasic monohydrate and sodium phosphate dibasic(S9638-25G, 57907-100G, Sigma-Aldrich), and (vi) 0.5 mM EDTA (BP120-500,Fisher Scientific) in water. The solutions were combined with 10 μl ofPCR reaction buffer. Luciferase amplification primers were 5′-GCC TGAAGT CTC TGA TTA AGT-3′ for the forward primer and 5′-ACA CCT GCG TCGAAG-3′ for the reverse primer, creating an amplicon of 96 bp [10].Amplification reactions were performed in a 20 μl solution containingMgCl₂ at 1.5 mM, primers at 0.2 μM (each), and 0.2 mM dNTPs in PCRbuffer and 0.025 units/μl of Taq polymerase (10966-034, Invitrogen) andSYBR-green (S-7563, Invitrogen) at 1:45,000. Aliquots of plasmid DNAwere diluted in water to generate a standard curve. Analysis wasperformed on iCycler PCR machine (Bio-Rad Laboratories, Inc.) usingoptical grade 96-well plates. Thermal cycle of the reaction was set asfollows: initial denaturation at 95° for 3 min, followed by 40 cycles ofdenaturation at 95° C. for 30-sec, 30-sec annealing at 60° C., and30-sec elongation at 72° C., all followed by a final extension of 10 minat 72° C. For each sample, three replicates were performed. For eachbuffer, the compatibility was calculated by comparing with the control(plasmid DNA in water).

FIG. 13 shows that sodium chloride, PBS, and sodium phosphate buffer wasincompatible as detection buffer for quantitative PCR assay as comparedwith control. However, use of tris-HCl or EDTA as buffer increased theanalytical assay's detection ability.

Example 8 Compatibility of LPMs With Nucleic-Acid-Based Tests

Compatibility of various LPMs (disclosed in EXAMPLE 1) with existingnucleic-acid-based tests was tested. Specifically, plasmid DNA was mixedwith different LPMs and the ability of qPCR to amplify DNA was assessed.LPMs were prepared by adding surfactants at various concentrations in 10mM Tris-HCl buffer. To mimic the process of tissue liquefaction asdisclosed in this text, each LPM was mixed with 0.2 mg/ml of pig skinhomogenate and ten million copies of Luciferase plasmid (E1741, PromegaCorp.) were spiked per 10 μl of LPM. This solution was combined with 10μl of PCR reaction buffer. qPCR was performed according to the protocoldescribed in Example 4. Purified plasmid DNA were diluted in a tris-HClsolution to generate a standard curve. Compatibility of each LPM wascalculated by detrmining the amount of plasmid amplified by qPCR andcomparing it with the control buffer (plasmid DNA in tris-HCl withoutsurfactant).

FIG. 14 shows that Triton X-100, Brij 30, DMSO, OTAB, OTAB-Brij 30, andDPS-Brij 30 were highly compatible with quantitative PCR; however LPMsconsisting of NLS or NLS-Brij30 failed to amplify the DNA. Notably,DPS-Brij 30 as a LPM effectively samples biomolecules from tissues,retains protein activity and is compatible with analytical methodsincluding ELISA, chromatography and qPCR. Therefore, DPS-Brij 30 is mostdesirable as liquefaction promoting media for analyzing proteins, lipidsand nucleic acids. Triton X-100 and DMSO, which have been known as afacilitator of PCR [11], were consistently shown to effectively producepolymerase-chain reactions; however, they do not yield satisfactorytissue extraction.

Example 9 Identification of Ultrasonic Parameters for Sampling Viableand Genetically-Intact Microorganisms From Tissues

This example describes a nonlethal condition of ultrasound toefficiently collect living microorganisms from tissues. Microorganismscan be collected from tissue by applying various form of energy totissues; however use of high energies is highly detrimental to theviability of microorganisms. Therefore, it is essential to find outnonlethal conditions of energy application for sampling livingmicroorganisms. We describe ultrasound exposure conditions for samplingviable and genetically-intact bacteria from skin.

Bacterial culture of E. Coli strain DH10α (18290-015, Invitrogen) weregrown in Luria-Bertani (BP1426, Fisher Scientific) at 37° C., 250 rpm oras solid culture on Agar plates (37° C.). Culture was harvested bycentrifugation and the resulting pellet was suspended in LPM comprisingof 10 mM Tris-HCl, pH 7.9 at a concentration of 10⁹ cells/ml. E. Colicells were quantified with a spectrophotometer (Biophotometer,Eppendorf), and a bacterial culture of 0.25×10⁹ cells/ml was consideredto correspond to an optical density absorbance value of 0.25 at awavelength of 600 nm. One ml of the resuspended cells was placed in asterilized cylindrical container (internal diameter 20 mm, flat base,1.3 mm wall thickness, 31 mm height). All experiments were performedwith a 600-Watt sonicator (Sonics & Materials, Newtown, Conn.) operatingat a frequency of 20 kHz at 50% duty cycle. The power setting and thetime of ultrasound exposure were varied in this experiment. Thetransducer was lowered into the container until the probe was immersedin the fluid at a distance of 5 mm from the bottom. The transducer wassterilized by 70% ethanol between sonication procedures on differentsamples. After sonication, 10-fold serial dilutions of each sample wereprepared in 10 mM Tris-HCl (pH 7.9). 100 ul of sample from each dilutionstep was plated onto Luria-Bertani agar and spread with a sterilespreader. The plates were incubated at 37° C. for 24 h and viablebacterial colony counts were made on the surface of agar plates. Resultswere expressed as percentage reduction in viability relative tonon-sonicated controls. To evaluate integrity of bacterial genome insamples exposed to ultrasound, electrophoresis was carried out. Allsamples were incubated at 56° C. in Proteinase K (19131, Qiagen) and0.5% (w/v) sodium lauryl sulfate (S529, Fisher Scientific). After 1-hincubation, total genomic DNA was extracted by using the DNeasy DNAExtraction Kit (69504, Qiagen). The standard protocol for the kit wasfollowed for all subsequent steps. The purified genomic DNA wasresuspended in 400 μl of Buffer AE and stored at −20° C. until analysis.The purified DNA was electrophoresed for 90 min at 100 V in a 2% (w/v)Tris-acetate-EDTA-agarose gel. The gels were stained with SYBR Gold (S11494, Invitrogen) and visualized under UV light.

FIG. 15 shows that viability of E. coli exposed to ultrasound at anintensity of 1.7 W/cm² for up to 2 min was statistically insignificantto the viability of non-treated E. coli samples. This suggests thatthese ultrasonic liquefaction conditions can be used for samplingbacteria without a major loss of viability. However, samples sonicatedat higher power output exhibited a more rapid decrease with applicationtime, and the cell viabilities were significantly different comparedwith non-treated cells. Even after 1 minute exposure at a higherintensity, viability was reduced to 3.6% (p<0.05). This observation isin agreement with bacterial genome integrity as assessed byelectrophoresis (FIG. 16). No damage to bacterial genome was observedupon sonication for 2 minutes at 1.7 W/cm² (conditions shown to maintaincell viability); however, in contrast, the genomic DNA of E. coli cellssonicated at intensities of 1.7 W/cm² (32% viability) and 2.4 W/cm² (8%viability) for 3 min were highly fragmented as can be seen by theirmigration to lower molecular weight part of the gel. These resultssuggest that collection of living bacteria should be performed at anultrasound intensity of 1.7 W/cm² for up to 2 min.

Example 10 Detection of Living Microorganisms from Tissues

A brief exposure of ultrasonic energy coupled with LPM (tris-HCl buffer)can sample viable bacteria from skin. Skin bacteria sampled byultrasound were quantified by the conventional colony counting assay aswell as real-time quantitative PCR, and evaluated by comparing withstandard sampling methods such as swabbing and the surfactant scrubbingtechnique.

In vitro experiments were performed on porcine skin to assess samplingof skin-resident bacteria. Pre-cut frozen full-thickness porcine skinharvested from the lateral abdominal region of Yorkshire pigs wasprocured in 10 cm×25 cm strips from Lampire Biological LaboratoriesInc., PA. The skin was stored at −70° C. until the experiment. Skinpieces with no visible imperfections such as scratches and abrasionswere thawed at room temperature and cut into small pieces (2.5 cm×2.5cm) and mounted on a Franz diffusion cell (Permegear, Hellertown, Pa.,USA). The receiver chamber of the diffusion cells was filled withphosphate buffered saline (PBS) (P4417, Sigma-Aldrich, St. Louis, Mo.)and the donor chamber (skin exposure area of 1.77 cm²) was filled with 1ml of 10 mM Tris-HCl buffer (pH 7.9), which also acted as the couplingfluid between the ultrasound transducer and skin. The ultrasoundtransducer was placed at a distance of 5 mm from the skin surface and anultrasonic intensity of 1.7 W/cm² was applied for 2 minutes. The probewas disinfected with 70% ethanol between experiments on differentsamples. As comparative controls, samples were obtained by swabbing theskin. Cotton swabs (B4320115, BD Diagnostics) were soaked in sterilizedphosphate-buffered saline before use. The area of the sample site wasstandardized by holding a sterilized metal ring enclosing an area of 3.3cm² onto the skin surface. The skin surface was rubbed gently andrepeatedly for approximately 20 seconds. Each swab was extracted with 1ml of PBS. Skin bacteria were also sampled by the surfactant scrubtechnique of Williamson and Kligman [2, 12]. A sterile metal ring wasfirmly held against the skin surface and 1 ml of 0.1% Triton X-100 in0.075 M phosphate buffer, pH 7.9, was pipette into it. The skin surfacewithin the ring was rubbed firmly for 1 min with a Teflon cell scraperand the resulting sample was collected in a sterile centrifuge-tube. Theprocedure was repeated at the same skin site for two additional timesand samples were pooled together. Serial 10-fold dilutions of eachsample were prepared and 100 μl aliquots from each diluted sample wereplaced on Tryptic Soy agar plates (90002, BD Diagnostics) [12]. Theplates were subsequently incubated under aerobic conditions at 37° C.for 24 hours and colonies were counted to obtain an estimate ofextraction efficiency by calculating the colony-forming unit per unitarea of sampled skin (CFU/cm²). To quantify total bacteria, real-timequantitative PCR was performed based on an amplicon of the 16S rRNAgene. All biological specimens were first incubated in a preparation ofenzymatic lysis buffer (20 mM Tris at pH 8.0, 2 mM EDTA, 1.2% TritonX-100) and lysozyme (20 mg/mL) for 30 min at 37° C. [9]. Subsequently,samples were incubated for 1 hour at 56° C. in Buffer AL and ProteinaseK from the DNeasy DNA Extraction Kit (Qiagen). The standard protocol forthe kit was followed for all subsequent steps. The DNA eluted by BufferAE was precipitated by incubation with equal volumes of absoluteisopropanol and then centrifuging for 20 min. The DNA pellets werewashed once with 70% ethanol, allowed to dry, and resuspended in 80 μlof Buffer AE. Negative controls were also prepared using untreatedsterile cotton swabs in PBS. Analysis of the 16S genes was performed onthe iCycler PCR machine (Bio-Rad Laboratories, Inc.) using optical grade96-well plates. A portion of the bacterial 16S gene was amplified usingforward primer 63F (5′-AGAGTTTGATCCTGGCTCAG-3′) and reverse primer 355R(5′-GACGGGCGGTGTGTRCA-3 [9, 13]. A standard curve was constructed byamplifying serial dilutions of genomic DNA from known quantities of E.Coli cells in 10 μl of Buffer AE. 10 μl of purified DNA was mixed with 2pmol of each primer and Platinum PCR Supermix (11784, Invitrogen) to afinal reaction volume of 20 μl Thermal cycling was set as follows:initial denaturation at 94° for 5 min, followed by 32 cycles of a 30-sec94° C. denaturation, 30-sec annealing at 66° C., and 30-sec elongationat 72° C., all followed by a final extension of 10 min at 72° C. Foreach sample, three replicates were performed.

FIG. 17 shows comparison of the sampling efficacies of differenttechniques. FIG. 17 a shows that ultrasonic sampling recoveredapproximately 17-fold higher number of bacteria from skin than cottonswabbing (p<0.05). Notably, counts of the total number of bacteriacollected by ultrasound did not differ significantly from the positivecontrol (surfactant scraping method). The effectiveness of ultrasonicsampling was further tested using quantitative real-time PCR based onamplifying the 16S rDNA bacterial gene (FIG. 17 b). Consistently,ultrasound collected 1.7×10⁴ bacteria/cm² which is significantly higherthan swabbing (4.5×10³ bacteria/cm²), and equivalent to scrubbingtechnique (1.6×10⁴ bacteria/cm²).

Example 11 Use of Sensitivity Enhancers to Facilitate Detection of HumanIgE in LPM

The ability of sensitivity enhancers to facilitate detection of a modelanalyte—human IgE antibody, which was dissolved in a model LPM—1% w/vNLS-Brij 30 in a PBS, was tested. ELISA assay was used to evaluatedetection of human IgE antibody in presence or absence of sensitivityenhancers in LPM. Specifically, 1 microgram of antibodies (A80-108A,Bethyl laboratory, TX) with specific binding to human IgE antibodies wascoated per well of a 96-well ELISA plate. Human IgE (RC80-108, Bethyllaboratory, TX) was dissolved in the LPM with or withour sensitivityenhancer at a concentration of 0-100 ng/ml. As a positive control, humanIgE samples were prepared by dissolving in a standard diluent containing1% w/v BSA and 0.05% w/v Tween 20 (P7949, Sigma-aldrich, MO) in 50 mMTris-buffered saline (T6664, Sigma-aldrich, MO) which is commonly usedin immunoassays. Two types of sensitivity enhancers were formulated: 10%BSA and 0.5% Tween 20 in PBS and 10% BSA and 0.5% Tween 20 in 50 mMTris-buffered saline. Each of these sensitivity enhancers was separatelyadded to LPM containing IgE in a ratio of 1:10. After 30-minutesincubation of ELISA plates with a standard blocking buffer, thesesamples were incubated in individual wells for 1 hour. After washing thewells, HRP-conjugated-secondary antibodies at a concentration of 1microgram/ml were incubated in each well for 1 hour. After washing, aHRP-based chemiluminescence signal (induced by substrates 54-61-00, KPL,MD), signifying detection ability of IgE antibodies by ELISA, wasmeasured for each test case using a spectrophotometer.

FIG. 18 plots the chemiluminescence signal intensity from various testcases as a function of analyte concentration. Results show that LPM byitself was not a suitable detection reagent for ELISA assay as comparedwith positive control. However, adding sensitivity enhancers to LPMincreased the analytical assay's detection ability. Additionally,tris-buffered saline was shown to elevate the signal intensity ascompared with phosphate-buffered saline, when they are used as solventsto prepare sensitivity enhancers. These results demonstrated that LPM byitself is not efficient in facilitating analyte detection by ELISA;however, addition of sensitivity enhancers can significantly enhancedetection ability of analytes by ELISA.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

Example 12 Delivery of Inulin and Acyclovir into Pig Skin

Drug delivery experiments were performed on pig skin in vitro. Pre-cutfrozen full-thickness porcine skin, harvested from the lateral abdominalregion of Yorkshire pigs, was obtained from Lampire BiologicalLaboratory, Inc., PA. The skin was stored at −80° C. freezer prior tothe experiment. The skin was thawed at room temperature, and the skinwith no visible imperfections such as scratches and abrasions were cutinto small pieces (2.5×2.5 cm). Skin pieces were mounted on to a Franzdiffusion cell (PermeGear, Inc., PA). Before each experiment, thereceiver compartment was filled with a LPM or phosphate buffer saline(PBS). A 1%-w/v mixture of NLS and Brij 30 in PBS was chosen as a modelformulation of a LPM. Prior to each experiment, the electricalconductivity of the skin was measured to ensure its integrity. The skinwas considered damaged if the initial conductivity was more than 2.2microA/cm². Ultrasound was applied using a sonicator (VCX 400, Sonicsand Materials) operating at a frequency of 20 kHz at an intensity of 2.4W/cm² for 5 minutes. After the LPM or PBS was removed, the donorcompartment was filled with 10 microCi/ml solution of Inulin(NET086L001MC, PerkinElimer Life and Analytical Sciences, Inc., MA) inPBS. Samples were taken from the receiver compartments 24 hours afterultrasound application. In a separate experiment, a rotating abrasivesurface (a circular brush with plastic bristles) was introduced in thedonor chamber such that it directly contacted the skin sample. 10microCi/m1 solution of Acyclovir was placed on the skin for 24 hours.The skin was washed by a saline and dissolved in Solvable (PerkinElmer,MA). The concentrations of those samples were measured by ascintillation counter (Tri-Carb 2100 TR, Packard, CT). All experimentswere conducted at room temperature, 22° C. Neither ultrasound norabrasive device was applied on the controls. Error bars indicate thestandard deviation.

5 minutes of ultrasound irradiation in combination with the LPMincreased drug transport, compared to that both by ultrasound alone andby the passive diffusion on intact skin, as shown in FIG. 19( a). Thesame effect was observed when the skin was abraded with a movingbrushing device comprising a plurality of bristles (FIG. 19( b)). Insummary, the examples using pig skin in vitro demonstrated that applyingenergy with a LPM is effective in enhancing the passage of moleculesthrough or into tissues. Parameters such as power, time of applicationand a formulation of a LPM can be optimized to suit the individualsituation, both with respect to the type of tissue and the substances tobe transported.

Although the present invention has been described in connection with thepreferred embodiments, it is to be understood that modifications andvariations may be utilized without departing from the principles andscope of the invention, as those skilled in the art will readilyunderstand. Accordingly, such modifications may be practiced within thescope of the following claims.

1-80. (canceled)
 81. A device for obtaining a liquefied sample from atissue, comprising: an energy source operatively connected to thetissue, wherein the energy source is not a heated liquid; a reservoirhousing operatively connected to the tissue; and a liquefactionpromoting medium, wherein the reservoir housing is configured to applythe liquefaction promoting medium to the tissue.
 82. The device of claim81, wherein the reservoir housing is configured to collect the liquefiedsample from the tissue.
 83. The device of claim 81, wherein theliquefaction promoting medium is contained within the reservoir housing.84. The device of claim 81, wherein the energy source is configured toapply an energy to the tissue, and wherein the energy is applied to thetissue within the reservoir housing.
 85. The device of claim 81, whereinthe reservoir housing comprises a sponge-bellow assembly, whichcomprises a sponge configured to at least one of: store the liquefactionpromoting medium, or collect the liquefied sample from the tissue. 86.The device of claim 81, further comprising a sample containeroperatively connected to the reservoir housing, wherein the samplecontainer is configured to contain at least one of: the liquefactionpromoting medium, and the liquefied sample.
 87. The device of claim 81,further comprising a suction pump operatively connected to the reservoirhousing, wherein the suction pump is configured to facilitate thecollection of the liquefied sample.
 88. The device of claim 81, whereinthe energy source is configured to apply an energy to the tissue, andthe energy is at least one of ultrasound energy, mechanical energy,optical energy, thermal energy, and electrical energy.
 89. The device ofclaim 81, wherein the energy source is configured to apply a mechanicalenergy to the tissue, and the mechanical energy is applied to the tissueby at least one of: a piezoelectric element, an abrasive component, avacuum, a pressure, and a shear force.
 90. The device of claim 81,wherein the energy source is configured to apply an optical energy tothe tissue, and the optical energy is applied to the tissue by a laser.91. The device of claim 81, wherein the energy source further comprisesan abrasive component connected to a shaft, and wherein the abrasivecomponent is at least one of: a sheet of abrasive material, an abrasivedisc, an abrasive ring, and a brush having bristles.
 92. The device ofclaim 81, wherein the energy source further comprises amicroneedle-based device comprising a plurality of microneedles.
 93. Thedevice of claim 81, wherein the reservoir housing includes a basesection, the device further comprising an abradable sheet positioned inthe base section of the reservoir housing.
 94. The device of claim 81,further comprising at least one of: a detection device operativelyconnected to the reservoir housing, and a diagnostic device operativelyconnected to the reservoir housing.
 95. The device of claim 81, furthercomprising an additional energy source, wherein the additional energysource is at least one of: an abrasive actuator, a mechanical motor, anelectro-magnetic actuator, a piezoelectric transducer, a suction device,and a pressure device.
 96. The device of claim 81, further comprising atleast one of: a catheter; and a diagnostic probe, wherein the diagnosticprobe is at least one of: an endoscope, a colonoscope, and alaparoscope.
 97. A device for obtaining a liquefied sample from atissue, comprising: an energy source operatively connected to thetissue, wherein the energy source is configured to apply a mechanicalenergy to the tissue; a reservoir housing operatively connected to thetissue; and a liquefaction promoting medium.
 98. The device of claim 97,wherein the reservoir housing is configured to at least one of; apply aliquefaction promoting medium to the tissue, and collect the liquefiedsample from the tissue.
 99. The device of claim 97, wherein themechanical energy is applied to the tissue by at least one of: apiezoelectric element, an abrasive component, a vacuum, a pressure, anda shear force.
 100. The device of claim 97, wherein the mechanicalenergy is applied to the tissue by an abrasive component connected to ashaft, and wherein the abrasive component is at least one of: a sheet ofabrasive material, an abrasive disc, an abrasive ring, and a brush withbristles.
 101. The device of claim 97, wherein the mechanical energy isapplied to the tissue by a microneedle-based device comprising aplurality of microneedles.
 102. A device for delivering a drug to aliquefied tissue, comprising: an energy source operatively connected tothe tissue; a reservoir housing operatively connected to the tissue; aliquefaction promoting medium; and a drug; wherein the reservoir housingis configured to apply to the tissue at least one of: the liquefactionpromoting medium, and the drug.
 103. The device of claim 102, whereinthe reservoir housing is configured to collect and analyze a sample ofthe liquefied tissue.
 104. The device of claim 102, wherein the energysource is configured to apply an energy to the tissue, and the energy isat least one of: ultrasound energy, mechanical energy, optical energy,thermal energy, and electrical energy.
 105. The device of claim 102,wherein the liquefaction promoting medium is configured to at least oneof: couple an energy from the energy source to the tissue, facilitateliquefaction of the tissue, store the drug to be delivered to thetissue, increase the solubility of the drug, and inhibit degradation ofthe drug.